MX2008007761A - Vaccine - Google Patents

Abstract

The present invention is in the field of pneumococcal capsular saccharide conjugate vaccines. Specifically, an immunogenic composition for infants is provided comprising a multivalent Streptococcus pneumoniae vaccine comprising 2 or more capsular saccharide conjugates from different serotypes, wherein the composition comprises a serotype 22F saccharide conjugate. Such a vaccine may be used in infant populations to reduce the incidence of elderly pneumococcal disease such as exacerbations of COPD and/or IPD.

Description

VACU NA FIELD OF THE INVENTION The present invention relates to an improved vaccine of Streptococcus pneumoniae Background of the invention Children less than 2 years of age do not have an immune response for most polysaccharide vaccines for what has been It is necessary to make immunogens to polysaccharides by chemical conjugation with a carrier protein. The coupling of the polysaccharide, a T-dependent antigen, to a protein, a T-dependent antigen, confers on the polysaccharide the T dependence properties, including isoty change, affi maturation and recall induction. However, there may be problems with the repeated administration of polysaccharide-protein conjugates, or with the combination of polysaccharide-protein conjugates to form multivalent vaccines. For example, it has been reported that a polysaccharide vaccine (PRP) against Haemophilus influenzae type b using tetanus toxoid (TT) as the carrier protein was tested in a dose range with simultaneous immunization with TT (free) and a pneumococcal vaccine of conjugate polysaccharide-TT following a standard children's program. As the dose of the pneumococcal vaccine increased, the immune response to the PRP polysaccharide portion of the Hlb conjugate vaccine decreased, which indicates immunological interference of the polysaccharide, possibly through the use of the same carrier protein (Dagan et al, Infect immun (1 998), 66: 2093-2098) It has also been proven that the effect of the carrier-protein dose on the Humoral response to the protein itself is multifaceted. In human infants, it was reported that increasing the dose of a tetravalent tetanus toxoid conjugate produced a diminished response to the tetanus carrier (Dagan et al., Supra). Classical analyzes of these effects of combination vaccines have been described as carrier-induced epitope suppression, which is not fully understood, but is believed to be the result of a surplus amount of carrier protein (Fattom, Vaccine 1 7: 1 26 ( 1999)). This seems to result in competition for Th cells, for B cells for the carrier protein, and for B cells for the polysaccharide. If the B cells for the carrier protein predominate, there are not enough Th cells available to provide the necessary help for the B cells specific for the polysaccharide. However, the immunological effects observed have been inconsistent, with the total amount of carrier protein in some cells. cases increasing the immune response, and in other cases decreasing the immune response. Therefore, the technical difficulties remain in combining multiple polysaccharide conjugates into a single effective vaccine formulation. Streptococcus pneumoniae is a Gram-positive bacterium responsible for considerable morbidity and mortality (particularly in young people of a certain age), producing invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonization, such as acute otitis media. It is estimated that the rate of pneumococcal pneumonia in the Ud States for people over 60 years of age is 3 to 8 per 1,000, 000. In 20% of cases, this leads to bacteremia, and other manifestations, such as meningitis, with a mortality rate close to 30% even when antibiotic treatment is used. The pneumococcus is encapsulated with a chemically bound polysaccharide that confers serotype specificity. There are 90 known serotypes of neu mococos, and the capsule the principle of virulence determined for pneumococci, since the capsule not only protects the inner surface of the complement bacteria, but by itself is poorly immunogenic. Polysaccharides are antigens independent of T, and can not be processed or presented in M HC molecules that interact with T cells. However, they can stimulate the immune system through an alternative mechanism that involves the cross-linking of surface receptors in B cells. In several experiments it was shown that protection against invasive pneumococcal disease is more strongly related to antibody specific to the capsule, and protection is serotype specific.

Streptococcus pneumoniae is the most common cause of bacterial disease and otitis media invasive in infants and young children. Similarly, older people have 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 Berk, (1983) Medicine (Baltimore) 62: 271-285]. The main clinical syndromes produced by S. pneumoniae are widely recognized and discussed in common medical textbooks (Fedson DS, Muscher DM En: Plotkin SA, Orenstein WA, editors, Vaccines, 4th edition, Philadelphia WB Saunders Co, 2004a 529 -588) For example, invasive pneumococcal disease (IPD) is defined as any infection in which S. pneumoniae is isolated from the blood or from 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 recognized because it comprises several conditions (obstruction of airflow, chronic bronchitis, bronchiolitis or small respiratory disease and emphysema) that often coexist. Patients experience exacerbations of their condition that are usually associated with shortness of breath, and often have an increased cough that can be caused by mucus or purulent sputum (Wilson, Eur Respir J 2001 17: 995-1007). The COPD Physiologically defined by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (American Society for the diagnosis and care of patients with chronic obstructive pulmonary disease.) Am J Respir Crit Care Med. 1995 Nov; 152 (5 Pt 2): S77-121). Exacerbations of COPD are often caused by bacterial infection (eg pneumococcal) (Sethi S, Murphy TF, Bacterial infection in chronic obstructive pulmonary disease 2000: a state-of-the-art review, Clin Microbiol Rev. 2001 Apr; 14 (2): 336-63). It is therefore an object of the present invention to develop an improved formulation of a polysaccharide conjugate vaccine against multiple serotype Streptococcus pneumoniae. Brief description of the figures Figure 1. Bar graph showing the immunogenicity of the 11-valent conjugate in elderly Rhesus monkeys. The lightest bars represent the GMC after two inoculations with 11-valent conjugate in aluminum phosphate adjuvant. The darker bars represent the GMC after two inoculations with 11-valent conjugate in adjuvant C. Figure 2. Bar graph showing the B memory cells for PS3 after inoculation with the 11-valent conjugate in adjuvant C or adjuvant aluminum phosphate. Figure 3. Bar graph showing 19F anti-polysaccharide immunogenicity in Balb / C mice for polysaccharides 4- simple valent and the 4-valent conjugates dPIy. Figure 4 Bar graph showing the anti-polysaccharide 22F immunogenicity in Balb / C mice for the simple 4-valent polysaccharides and the 4-valent phtD conjugates. Figure 5. Bar graph showing anti-22F IgG response in Balb / c mice. Figure 6. Bar graph showing anti-22F opsono-phagocytosis titers in Balb / c mice. Figure 7. Bar graph comparing IgG responses induced in young C57B1 mice after immunization with 13-valent conjugate vaccine formulated in different adjuvants.

Figure 8. Bar graph showing the protective efficacy of different combinations of vaccines in a monkey pneumonia model. Figure 9. Bar graph showing the anti-PhtD IgG response in Balb / c mice after immunization with 22F-PhtD or 22F-AH-PhtD conjugates. Figure 10. Protection against pneumococcal type 4 exposure in mice after immunization with 22F-PhtD or 22F-AH-PhtD. DESCRIPTION OF THE INVENTION The present invention provides an immunogenic composition for children that contains a multivalent vaccine of Streptococcus pneumoniae that contains 2 or more (for example 7, 8, 9, 10, 11, 12, 13, 14, 15) conjugated capsular saccharides of different serotypes, wherein the composition contains a conjugate saccharide 22F. Although infection in childhood by pneumococcal serotype 22F is not very common, the inventors believe that the presence of 22F in a pneumococcal infant vaccine will be collective immunity in the population in such a way that it can prevent the onset of a serious disease in age. advanced disease produced by this serotype (such as pneumonia and / or invasive pneumococcal disease (IPD) and / or exacerbations of chronic obstructive pulmonary disease (COPD)) or its severity can be reduced. For the purposes of this invention, "immunizing a human host against COPD exacerbations" or "treatment or prevention of COPD exacerbations" or "severity reduction of COPD exacerbations" refers to a reduction in the incidence or rate of COPD exacerbations. (for example, a reduction in the 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 example within a group of patients immunized with the compositions or vaccines of the invention. Thus, in one embodiment a method is provided to prevent an elderly human host from having a pneumococcal disease caused by infection with Streptococcus pneumoniae serotype 22F (or reducing its severity) which comprises administering to a human infantile host (or a human infantile population). ) an immunoprotective dose of the immunogenic composition or the vaccine of the invention. A use of the immunogenic composition or vaccine of the invention in the manufacture of a medicament for the prevention or reduction of severity of a disease caused by infection by Streptococcus pneumoniae serotype 22F in elderly human patients, where an immunoprotective dose of the composition or vaccine is administered to a human child (or child population). In one embodiment, the immunogenic composition contains capsule saccharide conjugates of Streptococcus pneumoniae from serogroups 19A and 19F, optionally wherein 19A is conjugated to a first bacterial toxoid and 19F is conjugated to a second bacterial toxoid. The term capsular saccharide includes polysaccharides and capsular oligosaccharides that can be obtained from the capsular polysaccharide. An oligosaccharide contains at least 4 sugar residues. The term bacterial toxoid includes bacterial toxins that are inactivated either by genetic mutation, by chemical treatment or by conjugation. Suitable bacterial toxoids include tetanus toxoid, diphtheria toxoid, pertussis toxoid, cytolysins or bacterial pneumolysins. Pneumolysin (PIy) mutations have been described that decrease the toxicity of pneumolysin (WO 90/06951, WO 99/03884). Similarly, genetic mutations of diphtheria toxin are known to decrease its toxicity (see below). Genetically detoxified diphtheria toxin analogs include CRM197 and other mutants described in US 4,709,017, US 5,843,711, US 5,601,827, and US. ,917,017. CRM197 is a non-toxic form of diphtheria toxin but is immunologically indistinguishable from diphtheria toxin. CRM197 is produced by C. diphtheriae infected by the non-toxigenic phase ß197tox- created by nitrosoguanidine mutagenesis of the toxigenic b carinophagus (Uchida et al. Nature New Biology (1971) 233; 8-11). The CRM197 protein has the same molecular weight as the diphtheria toxin, but differs from it in a single-base change in the structural gene. This leads to an amino acid change from glycine to glutamine at position 52, which makes fragment A unable to bind to NAD, and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem. 46; 69-94, Rappuoli Applied and Environmental Microbiology Sept 1983 p 560-564). The first and second bacterial toxoids may be the same or different. When the first and second bacterial toxoids are different, this means that they have a different amino acid sequence. For example, 19A and 19F can be conjugated with tetanus toxoid and tetanus toxoid; diphtheria toxoid and diphtheria toxoid; Crm197 and CRM197, pneumolysin and pneumolysin, tetanus toxoid and diphtheria toxoid; Tetanus toxoid and CRM197; Tetanus toxoid and pneumolysin; diphtheria toxoid and tetanus toxoid; diphtheria toxoid and CRM197, diphtheria toxoid and pneumolysin; CRM197 and tetanus toxoid, CRM197 and diphtheria toxoid; CRM197 and neumolisna; pneumolysin and tetanus toxoid; pneumolysin and diphtheria toxoid; or pneumolysin and CRM197, respectively.

In one embodiment, in addition to saccharide conjugate of S. pneumoniae 22F (and optionally 19A and 19F), the immunogenic composition further contains conjugates of capsular saccharides of S. pneumoniae 4, 6B, 9V, 14, 18C and 23F. In one embodiment, in addition to saccharide conjugate of S. pneumoniae 22F (and optionally 19A and 19F), the immunogenic composition also contains capsular saccharide conjugates of S. pneumoniae 1, 4, 5, 6B, 7Ft 9V, 14, 18C and 23F. In one embodiment, in addition to saccharide conjugate of S. pneumoniae 22F (and optionally 19A and 19F), the immunogenic composition further contains conjugates of capsular saccharide S. pneumoniae 1, 4, 5, 6B, 7F, 9V, 14, 18C, 22F and 23F. In one embodiment, in addition to S. pneumoniae conjugated saccharide of 22F (and optionally 19A and 19F), the immunogenic composition further contains conjugates of capsular saccharides of S. pneumoniae 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 22F and 23F. In one embodiment, in addition to saccharide conjugated S. pneumoniae 22F (and optionally 19A and 19F), the immunogenic composition further contains conjugates of capsular saccharide S. pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 22F and 23F. Commonly the Streptococcus pneumoniae vaccine of the present invention will contain capsular saccharide antigens (preferably conjugates), wherein the saccharides are derived from at least ten serotypes of S. pneumoniae. The quantity of capsular saccharides of S. pneumoniae can range from 10 serotypes different (or "V", valences) up to 23 different serotypes (23V). In one modality there are 10, 11, 12, 13, 14 or 15 different serotypes. In another embodiment of the invention, the vaccine may contain conjugated S. pneumoniae saccharides and unconjugated S. pneumoniae saccharides. Preferably, the total amount of saccharide serotypes is less than 23. For example, the invention may include 10 serotypes of conjugated and 13 unconjugated saccharides. Similarly, the vaccine may contain 11, 12, 13, 14 or 16 conjugated saccharides and 12, 11, 10, 9 or 7 respectively, unconjugated saccharides. In one embodiment, the multivalent pneumococcal vaccine of the invention will be selected from the following serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F , 18C, 19A, 19F, 20, 22F, 23F and 33F, although it will be taken into account that one or two serotypes could be substituted depending on the age of the recipient to receive the vaccine and the geographical location where it will be administered. the vaccine. For example, a 10-valent vaccine may contain polysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent vaccine may also include serotype 3 serotypes. A pediatric 12 or 13-valent (infantile) 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 15B, or 19A and 15B, or 22F and 15B, while a 13-valent vaccine for the elderly may include the 11-valent 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, .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 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 conjugates), for example capsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F are included. A further embodiment of the invention includes at least 12 or 13 saccharide antigens, for example a vaccine may contain capsular 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 other saccharide antigens, for example 23-valent (such as 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) are also contemplated by the invention. The vaccine of the present invention may contain protein D (PD) from Haemophilus influenzae (see for example EP 0594610). He Haemophilus influenzae is a key organism causing otitis media, and the inventors of the present have shown that including this protein in a Streptococcus pneumoniae vaccine will provide a level of protection against otitis media related to Haemophilus influenzae (reference publication POET). In one embodiment, the vaccine composition contains 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 as a carrier protein and as a free protein. Protein D can be used as a full-length protein or as a fragment (WO0056360). In a further aspect, protein D is present as a carrier protein for most saccharides, for example 6, 7, 8, 9 or more of the saccharides can be conjugated with protein D. In this aspect, protein D also it can be present as a free protein. The vaccine of the present invention contains one, two or more different types of carrier protein. Each type of carrier protein can act as a carrier for more than one saccharide, these saccharides can be the same or different. For example, serotypes 3 and 4 can be conjugated with the same carrier protein, either with the same carrier protein molecule or with different molecules of the same carrier protein. In one embodiment, two or more different saccharides can be conjugated with the same carrier protein, either with the same carrier protein molecule or with different molecules of the same carrier protein. Any capsular saccharides of Streptococcus pneumoniae present in the immunogenic composition of the invention can be conjugated to a carrier protein selected independently from the group consisting of TT, DT, CRM197, fragment C of TT, PhtD, PhtDE fusions (particularly those described in WO). 01/98334 and WO 03/54007), detoxified pneumolysin and protein D. A more complete list of carrier proteins that can be used in the conjugates of the invention is presented below. The carrier protein conjugate with one or more of the capsular saccharides of S. pneumoniae in the conjugates present in the immunogenic compositions of the invention is optionally a member of the polyhistidine (Pht) protein family, fragments or proteins. of merging them. The PhtA, PhtB, PhtD or PhtE proteins may have an amino acid sequence that shares 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with a sequence described in WO 00/37105 or WO 00/39299 (for example with amino acid sequence 1-838 or 21-838 of SEQ ID NO 4 of WO 00/37105 for PhtD). For example, the fusion proteins are composed of full-length proteins or fragments of 2, 3 or 4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins are PhWB, PhtA / D, PhWE, PhtB / A, PhtB / D, PhtB / E PhtD / A. PhtD / B, PhtD / E, PhtE / A, PhtE / B and PhtE / D, where the proteins are linked to the first mentioned in the term N (see for example WO01 / 98334). When fragments of Pht proteins are used (separately or as part of a fusion protein), each fragment optionally contains one or more histidine motifs in triad and / or coiled spiral regions of these polypeptides. A histidine motif in triad is the polypeptide part having the sequence HxxHxH, where H is histidine and x is an amino acid other than histidine. A threaded spiral region is a region predicted by the "Coils" algorithm. Lupus, A. et al (1 991) Science 252; 1 1 62-1 1 64. In one embodiment the fragment, or each fragment, includes one or more histidine motifs in triad, as well as at least one coiled spiral region. In one embodiment, the fragment, or each fragment, contains exactly or at least 2, 3, 4 or 5 histidine motifs in triad (optionally, with natural Pht sequence between the 2 or more triads, or intra-triads sequence that is more than 50, 60, 70, 80, 90 or 1 00% identical to a sequence natural pneumococcal intra-triad of Pht - for example the intra-triad sequence shown in SEQ ID NO 4 of WO 00/371 05 for PhtD). In one embodiment, the fragment, or each fragment, contains exactly or at least 2, 3 or 4 coiled spiral regions. In one embodiment, a Pht protein described herein includes the full length protein with the bound signal sequence, the full-length mature protein with the removed signal peptide (e.g. amino acids in the term N), natural origin variants of Pht protein and immunogenic fragments of Pht protein (for example fragments as described above or poly peptides containing at least 1 5 or 20 contiguous amino acids of an amino acid sequence) in WO00 / 371 05 or WO00 / 39299 wherein said polypeptide is capable of eliciting a specific immune response for said amino acid sequence in WO00 / 371 05 or WO00 / 39299). In particular, the term "PhtD" as used herein includes the full-length protein with the bound signal sequence, the full-length mature protein with the signal peptide removed (e.g., 20 amino acids in the N-terminus), naturally occurring variants of PhtD and immunogenic fragments of PhtD (eg fragments as described above or polypeptides containing at least 15 or 20 contiguous amino acids of a PhtD amino acid sequence in WO00 / 371 05 or WO00 / 39299 wherein said polypeptide is capable of eliciting a specific immune response for said PhtD amino acid sequence in WO00 / 371 05 or WO00 / 39299 (for example S EQ IDNO: 4 of WO 00/371 05 for PhtD) If the carrier protein is the same for 2 or more saccharides in the composition, the saccharides could be conjugated with the same molecule of the carrier protein (carrier molecules that have 2 different saccharides plus conjugates with it s) [see for example WO 04/083251]. Alternatively, the saccharides can be each conjugated separately with different molecules of the carrier protein (each molecule of carrier protein only has one type of saccharide conjugate for it). Examples of carrier proteins that can be used in the present invention are DT (diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, DT CRM197 (a mutant of DT) other point mutants of DT, 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, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and / or Ala 158 for Gly and other mutations described 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 described in US 5917017 or US 6455673; or fragment described in US 5843711, pneumococcal pneumolysin (Kuo et al (1995) Infecí Immun 63; 2706-13) including ply detoxified in some form, for example dPLY-GMBS (WO 04081515, PCT / EP2005 / 010258) or dPLY-formaldehyde , 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 AE are described in more detail below) OMPC (outer membrane meningococcal protein - usually extracted from N. meningitidis serogroup B - EP0372501), PorB (from N. meningitidis), PD (protein D from Haemophilus influenzae - see, for example, EP 0 594 610 B), or their equivalents immunologically functional peptides, 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 containing multiple epitopes of human CD4 + T cells of various antigens derived from pathogens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al (2004) Infecí Immun 72; 4884-7) pneumococcal surface protein PspA (WO 02/091998), iron absorption proieins (WO 01/72337), loxin A or B of C. difficile (WO 00/61761). Nurkka et al. Pediatric Infectious Disease Journal. 23 (11): 1008-14, 2004 Nov. described an 11-valent pneumococcal vaccine with all serotypes conjugated with PD. However, prevalence rates have shown that opsonophagocytic activity improved for antibodies induced with conjugates containing 19F conjugated with DT compared to 19F conjugated with PD. further, the invention of the présenle have demonstrated that a greater cross reactivity is observed for 19A with 19F conjugated with DT. It is therefore a characteristic of the composition of the present invention that serotype 19F is conjugated with a bacterial toxoid, for example TT, pneumolysin, DT or CRM 197. In one aspect, serotype 19F is conjugated with DT. It is also a feature of the invention that serotype 19A is conjugated to a bacterial toxoid, for example TT, pneumolysin, DT or CRM 197. The remaining saccharide serotypes of the immunogenic composition sludges may be conjugated to one or more carrier proteins other than DT (ie, only 19F is conjugated to DT), or may be divided among one or more carrier proteins other than DT and DT alone. In one modality, 19F is conjugated with DT or CRM 197 and all remaining serotypes are conjugated with PD. In a further embodiment, 19F is conjugated to DT or CRM 197, and the remaining serotypes are divided 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 conjugate with TT. In one aspect of this embodiment, said 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 divided 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 divided. between PD, TT and pneumolysin. In a further embodiment, 19F is conjugated to DT or CRM 197, and the remaining serotypes are divided among PD, TT and CRM 197. In a further embodiment, 19F is conjugated to DT or CRM197 and the remaining serotypes are divided among 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 and the remaining serotypes are divided among PD, TT, pneumolysin and optionally PhtD or fusion protein PhtD / E. In an additional modality, 19F is conjugated with DT or CRM197, 19A is conjugated with pneumolysin or TT, an additional saccharide, is conjugated with TT, another saccharide is conjugated with PhtD or PhtD / E and all the other saccharides are conjugated with PD. In a further embodiment, 19F is conjugated to DT or CRM197, 19A is conjugated to pneumolysin, another saccharide is conjugated to TT, another saccharide is conjugated to pneumolysin, 2 additional saccharides are conjugated to PhtD or PhtD / E and all additional saccharides are conjugated with PD. In one embodiment, the immunogenic composition of the invention contains Protein D of Haemophilus influenzae. Within this modality, If PD is not one of the carrier proteins used to conjugate any other saccharides other than 19F, for example, 19F is conjugated to DT, while the other serotypes are conjugated to one or more different carrier proteins, which they are not PD, then the PD will be present in the vaccine composition as a free protein. If PD is one of the carrier proteins used for saccharide conjugates other than 19F, then PD may optionally be present in the vaccine composition as a free protein. The term "saccharide" in this specification may indicate polysaccharide or oligosaccharide and includes both. The polysaccharides are isolated from bacteria and can be sized to some extent by known methods (see for example EP497524 and EP497525) and preferably by microfluidization. The polysaccharides can be dimensioned in order to reduce the viscosity of the samples of polysaccharide and / or to improve the filterability of the conjugated products. Oligosaccharides have a low amount of repeating units (commonly 5-30 repeating units) and are commonly hydrolyzed polysaccharides. The capsular polysaccharides of Streptococcus pneumoniae include repeating oligosaccharide units which may contain up to 8 sugar residues. For a review of the oligosaccharide units for the key serotypes of Streptococcus pneumoniae see JON ES, Christopher. Vaccines based on the cell l its rface carbohyd rates of pathogen ic bacteria. An. Acad. Bras. Cieñe, 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 an oligosaccharide unit, or one shorter than the natural length saccharide chain of oligosaccharide repeat units. In one embodiment, all saccharides present in the vaccine are polysaccharides. The full-length polysaccharides can be "sized", that is, their size can be reduced, by various methods, such as acid hydrolysis treatment, hydrogen peroxide treatment, emulsiflex® mediation followed by peroxide treatment. hydrogen to generate olosaccharide fragments or microfluidization. The inventors have also noted that the approach of the art has been to use oligosaccharides to facilitate production of the conjugated The inventors have found that by using natural or slightly sized polysaccharide conjugates, one or more of the following advantages can be obtained: 1) a conjugate having a high degree of immunogenicity, which is filterable, 2) the ratio of polysaccharide to protein in the conjugate can be altered in such a way that the ratio of polysaccharide to protein (w / w) in the conjugate can be increased (which can have an effect on the deletion effect of the carrier), 3) the conjugates inm Unigenes prone to hydrolysis can be stabilized by the use of larger saccharides for conjugation. The use of larger polysaccharides can result in more cross-linking with the carrier conjugate and can decrease the release of the free saccharide from the conjugate. The conjugate vaccines described in the prior art tend to depolymerize the polysaccharides before conjugation in order to improve conjugation. The inventors of the present invention have found that saccharide conjugate vaccines that retain a larger saccharide size can provide a good immune response against pneumococcal disease. The immunogenic composition of the invention can then contain one or more saccharide conjugates, wherein the average size (eg weight average molecular weight; Mw) of each saccharide before conjugation is greater than 80 kDa, 1 00 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa or 1 000 kDa. In one embodiment, one or more saccharide conjugates of the invention must have an average saccharide size before conjugation, of 50-1 600, 80-1 400, 1 00-1 000, 1 50-500, or 200-400 kDa (note that when the average size is Mw, the units 'kDa' will be replaced here by 'x1 03'). In one embodiment, the conjugate after conjugation must be easily filterable through a 0.2 micron filter, in such a way that a production of more than 50, 60, 70, 80, 90 or 95% is obtained after filtration compared to the sample before filtration. For the purposes of the invention, "natural polysaccharide" refers to a saccharide that has not been subjected to a process (eg post-purification), whose purpose is to reduce the size of the saccharide. A polysaccharide may become slightly red in size during normal purification procedures. This type of saccharide is still natural. Only if the polysaccharide has been subjected to sizing techniques does the polysaccharide not be considered natural. For the purposes of the invention, "sized by a factor of up to x2" means that the saccharide is subjected to a process aimed at reducing the size of the saccharide but to maintain a size of more than half the size of the natural polysaccharide. X3, x4 etc. they must be interpreted in the same way, that is, the saccharide is subject to a process designed to reduce the size of the polysaccharide, but it maintains a size of more than a third, a quarter, etc. of the size of the natural polysaccharide. In one aspect of the invention, the immunogenic composition contains Streptococcus pneumoniae saccharides of at least 10 serotypes conjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or each saccharide of S. pneumoniae is a natural polysaccharide. In one aspect of the invention, the immunogenic composition contains Streptococcus pneumoniae saccharides of at least 10 serotypes conjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or each saccharide of S. pneumoniae is sized by a factor of up to x2, x3, x4, x5, x6, x7, x8, x9 or x10. In one embodiment of this aspect, the majority of the saccharides, for example 6, 7, 8 or more of the saccharides are sized by a factor of up to x2, x3, x4, x5, x6, x7, x8, x9 or x10. The molecular weight or average molecular weight (or size) of a saccharide of the present refers to the weight average molecular weight (Mw) of the saccharide measured before conjugation and is measured by MALLS. The MALLS technique is well known in the art and is commonly carried out as described in example 2. For the MALLS analysis of pneumococcal saccharides, two columns (TSKG6000 and 5000PWxl) can be used in combination and the saccharides are eluted in water. The saccharides are detected using a light scattering detector (e.g. Wyatt Dawn DSP equipped with an argon laser of 10 mW at 488 nm) and a thermometric refractometer (e.g. Wyatt Otilab DSP equipped with a P1000 cell and a red filter at 498 nm). ).

In one embodiment the saccharides of S. pneumoniae are natural polysaccharides or natural polysaccharides that have been reduced in size during a normal extraction process. In one embodiment, S. pneumoniae saccharides are sized by mechanical division, for example by microfluidization or sound treatment. Microfluidization and sound treatment have the advantage of decreasing the size of the larger natural polysaccharides sufficiently to provide a filterable conjugate. The sizing is by a factor of no more than x20, x10, x8, x6, x5, x4, x3 or x2. In one embodiment, the immunogenic composition contains Conjugates of S. pneumoniae which are made of a mixture of natural polysaccharides and saccharides which are sized by a factor of not more than x20. In one aspect of this embodiment, the majority of the 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 one embodiment, the saccharide of Streptococcus pneumoniae is conjugated to the carrier protein by a linker, for example a bifunctional linker. The binder is optionally heterobifunctional or homobifunctional, for example it has a reactive amino group and a reactive carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups. The binder for example has between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possible linker is ADH. Other binders include B-propionamide (WO 00/10599), 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 one embodiment, ADH is used as a linker to conjugate saccharides of serotype 18C. In one embodiment, ADH is used as a linker to conjugate saccharides of serotype 22F. The saccharide conjugates present in the immunogenic compositions of the invention can be prepared by any known coupling technique. The conjugation method can be supported by the activation of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide can be coupled in this way directly or by means of a separating group (linker) to an amino group in the carrier protein. For example, the spacer could be cystamine or cysteamine to give a thiolated polysaccharide that could be coupled to the carrier by means of a thioether linkage obtained after the reaction with a carrier protein activated with maleimide (for example using GMBS) or a haloacetylated carrier protein. (for example using iodoacetimide [for example, ethyl iodoacetimide, HCl] or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or ADH and the amino derivative saccharide is conjugated to the carrier protein using carbodiimide chemistry (for example EDAC or EDC) by means of a carboxyl group on the protein carrier These conjugates are described in published PCT application WO 93/15760 Uniformed Services University and WO 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. The conjugation may involve a carbonyl binder which can be formed by reaction of a free hydroxyl group of the saccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4, Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction with a protein to form a carbamate linkage. This may involve reduction of the anomeric term to a primary hydroxyl group, optional protection / deprotection of the reaction of the primary hydroxyl group, the primary hydroxyl group with CDI to form a carbamate intermediate product CDI and coupling the intermediate product carbamate CDI with an amino group in a protein. The conjugates can also be prepared by reductive amination methods as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508. A further method involves the coupling of an activated saccharide with cyanogen bromide (or CDAP) derivative with adipic acid dihydrazide (ADH) for the carrier protein by means of carbodiimide condensation (Chu C. et al., Immunity, 1983245256), for example. using EDAC.

In one embodiment, a hydroxyl group (preferably an activated hydroxyl group, for example an activated hydroxyl group to make a cyanate ester [eg, with CDAP]) in a saccharide, is linked to an amino or carboxylic group in a protein either straight or indirectly (through a linker). When a binder is present, a hydroxyl group in a saccharide is preferably linked to an amino group in a binder, for example using CDAP conjugation. Another amino group in the linker, for example ADH) can be conjugated to a carboxylic acid group in a protein, for example using carbodiimide q, for example using E DAC. In one modality, the saccharide or pneumococcal capsular saccharides are conjugated with the binder first, before the binder is conjugated with the carrier protein. Alternatively, the binder can be conjugated with the carrier prior to conjugation with the saccharide. A combination of techniques can also be used, with some saccharide-protein conjugates prepared by CDAP, and some by reductive amination. In general, the following types of chemical groups can be used in a carrier protein can for coupling / conjugation: A) Carboxyl (for example, by aspartic acid or glutamic acid). In one embodiment, this group is linked to amino groups in saccharides directly or to an amino group in a linker with a carbodiimide chemical, for example, with E DAC.

B) Amino group (for example, by lysis). In one embodiment this group is linked to carboxyl groups in saccharides directly or to a carboxyl group in a linker with carbodiimide q, for example EDAC. In another embodiment this group is linked to hydroxyl groups activated with CDAP or CN Br in saccharides directly or to these groups in a linker; to saccharides or binders that have an aldehyde group; to saccharides or linkers having a succinimide ester group. C) Sulfhydryl (for example, by cysteine). In one embodiment, this group is linked to a bromo sacral or acetylated chlorine or a linker with maleimide chemical. In one embodiment this group is activated / modified with bis diazobenzid ina. D) Hydroxyl group (for example, by tyrosine). In one embodiment this group is activated / modified with bis diazobenzid ina. E) Imidazolyl group (for example, by histidine). In one embodiment, this group is activated / modified with bis diazobenzidine.

F) Guanidyl group (for example, by arginine). G) Indolyl group (for example, by tryptophan). In a saccharide, in general the following groups can be used for a coupling: OH, COOH or N H2. It is possible to generate aldehyde g rutes after different treatments known in the art, such as: periodate, acid hydrolysis, hydrogen peroxide, etc. Direct coupling approaches: Saccharide-OH + CM Br or CDAP - > cyanate ester + N H2-Prot - > conjugate Saccharide-aldehyde + NH2-Prot - > Schiff Base + NaCNBH3 - > conjugate Saccharide-COOH + NH2-Prot + EDAC - > conjugate Saccharide-NH2 + COOH-Prot + EDAC - > conjugate indirect collection approaches by separator (linker): Saccharide-OH + CNBr or CDAP - > cyanate ester + NH2 - NH2 - > saccharide - NH2 + COOH-Prot + EDAC - > conjugate Saccharide-OH + CNBr or CDAP - > cyanate ester + NH2 - SH - > saccharide - SH + SH-Prot (natural protein with a cysteine exposed or obtained after modification of amino groups of the protein by SPDP for example) - > Saccharide-S-S-Prot 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 + haloacetylated-Prot - > Saccharide-COOH + EDAC + NH2 NH2 - > saccharide NH2 + EDAC + COOH- Prot - > conjugate Saccharide-COOH + EDAC + NH2-SH- > saccharide - SH + SH- Prot (natural protein with a cysteine exposed or obtained after modification of amino groups of the protein by SPDP for example) - > Saccharide-S-S-Prot Saccharide-COOH + EDAC + NH 2 -SH - > saccharide - SH + maleimide-Prot (modification of amino groups) - > conjugate Saccharide-COOH + EDAC + NH 2 -SH - > Saccharide-SH + haloacetylated-Prot - > Saccharide-Aldehyde Conjugate + NH2-NH2 - > saccharide - NH2 + EDAC + COOH-Prot - > conjugate Note that instead of EDAC in the above, any carbodiimide can be used. In summary, the types of chemical carrier protein group that can be used in general for coupling with a saccharide are amino groups (for example in lysine residues), COOH groups (for example in residues of aspartic acid and glutamic acid) and SH groups (if they are accessible) (for example in cysteine residues). Preferably the ratio of carrier protein to saccharide of S. pneumoniae is between 1: 5 and 5: 1; for example, between 1: 0.5-4: 1, 1: 1-3.5: 1, 1.2: 1-3: 1, 1.5: 1-2.5: 1; for example, between 1: 2 and 2.5: 1; 1: 1 and 2: 1 (p / p). In one embodiment, the majority of conjugates, for example 6, 7, 8, 9 or more of the conjugates have a carrier protein to saccharide ratio that is greater than 1: 1, for example 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1 or 1.6: 1. In one embodiment, at least one S. pneumoniae saccharide is conjugated to a carrier protein by a linker using CDAP and EDAC. For example, 18C or 22F can be conjugated to a protein by a linker (for example those having two hydrazino groups at their ends, such as ADH) using CDAP and EDAC as described above. When use a binder, CDAP can be used to conjugate the saccharide with a binder, and then EDAC can be used to conjugate the binder with a protein or, alternatively EDAC can be used before conjugating the binder with the protein, after which the CDAP can be used to conjugate the binder with the saccharide. In general, the immunogenic composition of the invention may contain a dose of each saccharide conjugate of between 0.1 and 20 μg, 1 and 10 μg or 1 and 3 μg of saccharide. In one embodiment, the immunogenic composition of the invention contains each capsular saccharide of S. pneumoniae in a dose of between 0.1-20 μg; 0.5-10 μg; 0.5-5 μg or 1-3 μg of saccharide. In one embodiment, capsular saccharides may be present in different doses, for example some capsular saccharides may be present in a dose of exactly 1 μg or some capsular saccharides may be present in exactly 3 μg dose. In one embodiment, the saccharides of serotypes 3, 18C and 19F (or 4, 18C and 19F) are present in a er dose than other saccharides. In one aspect of this embodiment, serotypes 3, 18C and 19F (or 4, 18C and 19F) are present in a dose of about or exactly 3 μg, while other saccharides in the immunogenic composition are present in a dose of about or exactly 1 μg. "Around" or "approximately" are defined as within 10% or so of the figure given for the purposes of the invention In one embodiment, at least one of the capsular saccharides of S. pneumoniae is directly conjugated to a carrier protein (for example using one of the chemicals described above); Preferably the at least one of the capsular saccharides of S. pneumoniae is directly conjugated by CDAP. In one embodiment, the majority of capsular saccharides for example 5, 6, 7, 8, 9 or more are linked directly to the carrier protein by CDAP (see WO 95/08348 and WO 96/29094). The immunogenic composition may contain Streptococcus pneumoniae proteins, referred to herein as Streptococcus pneumoniae proteins of the invention. These proteins can be used as carrier proteins, or they can be present as free proteins, or they can be present as carrier proteins and as free proteins. The Streptococcus pneumoniae proteins of the invention are exposed to the surface, at least during part of the life cycle of the pneumococcus,. Preferably the proteins of the invention are selected from the following categories, such as proteins having a type II signal sequence motif of LXXC (wherein X is any amino acid, for example, the family of polyhistidine in triad (PhtX)). ), choline-binding proteins (CbpX), proteins having a type I signal sequence motif (e.g., Sp1 01), proteins having an LPXTG motif (where X is any amino acid, example, Sp128, Sp130), and toxins (e.g., PIy). Preferred examples within these categories (or motifs) are the following proteins, or their immunologically functional equivalents. In one embodiment, the immunogenic composition of the invention contains at least 1 protein selected from the group consisting of the Poly Histidine family in triad (PhtX), choline binding protein family (CbpX), CbpX truncates, LytX family, LytX truncates, proteins chimeric truncated CbpX-truncated LytX (or fusions), pneumolysin (PIy), PspA, PsaA, Sp128, Sp101, Sp130, Sp125 and Sp133. In a further embodiment, the immunogenic composition contains 2 or more proteins selected from the group consisting of the polyhistidine family in triad (PhtX), the choline binding protein family (CbpX), CbpX truncates, LytX family, LytX truncates, chimeric proteins truncated CbpX-truncated LytX (or fusions), pneumolysin (PIy), PspA, PsaA, and Sp128. In a further embodiment, the immunogenic composition contains 2 or more proteins selected from the group consisting of the poly triamine family in triad (PhtX), choline binding protein family (CbpX), CbpX truncates, LytX family, LytX truncates, chimeric proteins of truncated CbpX-truncated LytX (or fusions), pneumolysin (PIy), and Sp128. The Pht family (Poly Histidine Triad) contains PhtA, PhtB, PhtD, and PhtE proteins. The family is characterized by a lipidation sequence, two domains separated by a proline-rich region and several triads of histidine possibly involved in metal or nucleoside binding or enzymatic activity, (3-5) regions of spiral wound, a conserved N term and a heterogeneous C term. This is present in all strains of pneumococci analyzed. Homologous proteins were also found in other streptococci and in Neisseria. In one embodiment of the invention, the Pht protein of the invention is PhtD. However, it is understood that the terms Pht A, B, D, and E refer to proteins having the sequences described in what is cited below, as well as variants of them of natural origin (and elaborated by man) which have a sequence homology that is at least 90% identical to that of the referred proteins. Preferably this is at least 95% identical, and much more preferably it is 97% identical. With respect to PhtX proteins, PhtA is described in WO 98/18930, and is also referred to as Sp36. As indicated in the above, this is a protein of the polyhistidine family in triad and has the type II signal motif of LXXC. PhtD is described in WO 00/37105, and is also referred to as SpO36D. As indicated in the above, it is also a protein of the polyhistidine family in triad and has the type II signal motif LXXC. PhtB is described in WO 00/37105, and is also referred to as SpO36B. Another member of the PhtB family is the polypeptide that is degraded with C3, as described in WO 00/17370. This protein is also of the poly-histidine family in triad and has the type II signal motif LXXC. A preferred immunologically functional equivalent is the Sp42 protein described in WO 98/18930. A truncate of PhtB (approximately 79kD) is described in WO99 / 15675, which is also considered as a member of the PhtX family. PhtE is described in WO00 / 30299 and is called BVH-3. When referring to any Pht protein here, 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 of any Pht protein. A reference to PhtD or PhtB is also a reference to PhtDE or PhtBE fusions as found, for example, in WO0198334. Pneumolysin is a multifunctional toxin with an activation of cytolytic (hemolytic) and different complement activities (Rubins et al Am Respi Cit Care Med, 153.1339-1346 (1996)). The toxin is not secreted by pneumococci, but is released upon the lysis of pneumococci under the influence of autolysin. Its effects include, for example, the stimulation of the production of inflammatory cytokines by human monocytes, the inhibition of the pulsation of the cilia in the human respiratory epithelium, and the decrease of the bacterial activity and the migration of neutrophils. The most obvious effect of pneumolysin is in the lysis of red blood cells, which involves fixation to cholesterol. Because it is a toxin, it has to be detoxified (that is, it is non-toxic to a human being when administered at an appropriate dose for protection) before this, it can be administered in vivo. The expression and cloning of native or native type pneumolysin is known in the art. See, for example, Waiker et al. (Infect Immun, 55: 1 184-1 189 (1987)), Mitchell et al. (Biochim Biophys Acta, 1007: 67-72 (1989) and Mitchell et al (NAR, 18: 4010 (1990)). Detoxification of ply can be done by chemical means, for example, undergoing treatment with formalin or glutaraldehyde or a combination of both (WO 04081515, PCT / EP2005 / 010258) These methods are well known in the art for various toxins Alternatively, the ply can be genetically detoxified Thus, the invention comprises derivatives of pneumococcal proteins which can be example, mutated proteins The term "mutated" is used herein to indicate a molecule that 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. described in the foregoing, a ply mutant protein can be altered so that it is biologically inactive while still maintaining its immunogenic epitopes, see, for example, WO90 / 06951, B erry er al. (Infect Immun, 67: 981 -985 (1999)) and WO99 / 03884. As used herein, it is understood that the term "PIy" refers to mutated or detoxified pneumolysin appropriate for medical (ie, non-toxic) use. Regarding the choline binding protein family (CbpX), members of that family were originally identified as pneumococcal proteins that could be purified by choline affinity chromatography. All other choline binding proteins are not covalently bound to portions Phosphorylcholine of cell wall teichoic acid and lipoteichoic acid associated with membrane. Structurally, they have several regions in common over the entire family, although the exact nature of the proteins (sequence of amino acids, length, etc.) may vary. In general, the choline binding proteins contain an N (N) terminal region, conserved repeat regions (R1 and / or R2), a proline-rich region (P) and a conserved choline binding region ( C), elaborated of multiple repetitions, that constitutes approximately half of the protein. As used in this application, the term "Choline binding protein family (CbpX)" is selected from the group consisting of the choline binding proteins identified in WO97 / 41 1 51, PbcA, SpsA, PspC, CbpA, CbpD , and CbpG. CbpA is described in WO97 / 41 1 51. CbpD and CbpG are described in WO00 / 29434. PspC is described in WO97 / 09994. PbcA is described in WO98 / 21 337. SpsA is a choline binding protein described in WO 98/39450. Preferably the choline binding proteins are selected from the group consisting of CbpA, PbcA, SpsA and PspC. Another preferred embodiment is truncated CbpX wherein "CbpX" is as defined above and "truncated" refers to CbpX proteins that lack 50% or more of the binding region to Choline (C). Preferably these proteins lack the complete choline binding region. More preferably, said protein truncates lack (i) the choline binding region and (i) a part of the N-terminal half of the protein as well, which still maintains at least one repetition region (R1 or R2). More preferably still, the truncation has 2 repeat regions (R1 and R2). Examples of these preferred embodiments are NR1xR2 and R1xR2 as illustrated in WO99 / 51266 or WO99 / 51 188, 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 proteins associated with cell lysis. The terminal N domain comprises domain or choline binding domains, however the LytX family does not have all the characteristics found in the CbpA family indicated in the foregoing and therefore, for the present invention, the LytX family is considered different of the CbpX family. In contrast to the CbpX family, the terminal C domain contains the catalytic domain of the LytX protein family. The family contains LytA, B and C. With respect to the LytX family, LytA is described in Ronda et al., Eur J Biochem, 164: 621-624 (1987). LytB is described in WO 98/18930, and is also referred to as Sp46. LytC is also described in WO 98/18930, and is also referred to as Sp91. A preferred member of that family is LytC. Another preferred embodiment is the LytX truncates, where "LytX" is as defined above and "truncated" refers to LytX proteins that lack 50% or more of the choline binding region. Preferably these proteins lack the complete choline binding region. Still another preferred modality of this invention are truncated chimeric CbpX-truncated LytX proteins (or fusions). Preferably they contain NR1xR2 (or R1xR2) of CbpX and the C-terminal part (Cterm, ie, lacking the choline binding domains) of LytX (e.g., LytCCterm or Sp91Cterm). More preferably, CbpX is selected from the group consisting of CbpA, PbcA, SpsA and PspC. Even more preferably, this is CbpA. Preferably, the LytX is LytC (also called Sp91). Another embodiment of the present invention is a truncated PspA or PsaA that lacks the choline binding domain (C) and is expressed as a fusion protein with LytX. Preferably, LytX is LytC. With respect to PsaA and PspA, both are known in the art. For example, PsaA and its transmembrane deletion variants have been described by Berry and Patón, Infecí Immun 1996 Dec; 64 (12): 5255-62. PspA and its transmembrane deletion variants have been described, for example, in US 5804193, WO 92/14488, and WO 99/53940. Sp128 and Sp130 are described in WO00 / 76540. Sp125 is an example of a surface pneumococcal prolein with the molium anchored to the cell wall of LPXTG (where X is any amino acid). Any protein within this class of pneumococcal surface protein with such a motif has been found to be useful in the context of this invention, and therefore is considered another protein of the invention. The Sp125 itself is described in WO 98/18930, and is also known as ZmpB - a zinc metalloproteinase. Sp101 is described in WO 98/06734 (where it has the reference number y85993). It is characterized by a type I signal sequence. Sp133 is described in WO 98/06734 (where it has the reference number y85992). It is also characterized by a lipo I signal sequence. Examples of preferred prolein anligens of Moraxelia catarrhalis which can be included in a combination vaccine (especially for the prevention of middle oliis) are: OMP106 [WO 97/41731 (Anlex) and WO 96/34960 (PMC)]; OMP21 or fragments thereof (WO 0018910); LbpA and / or LbpB [WO 98/55606 (PMC)]; TbpA and / or TbpB [WO 97/13785 and WO 97/32980 (PMC)]; CopB [Helminen ME, et al. (1993) Infecí, Immun. 61: 2003-2010]; UspA1 and / or UspA2 [WO 93/03761 (Universiíy of Texas)]; OmpCD; HasR (PCT / EP99 / 03824); PilQ (PCT / EP99 / 03823); OMP85 (PCT / EPOO / 01468); Iipo06 (GB 9917977.2); Lipol O (GB 9918208.1); lipol 1 (GB 9918302.2); lipol 8 (GB 9918038.2); P6 (PCT / EP99 / 03038); D15 (PCT / EP99 / 03822); OmplAI (PCT / EP99 / 06781); Hly3 (PCT / EP99 / 03257); and OmpE. Examples of Haemophilus influenzae anigenes or non-classifiable fragments thereof that 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 containing peptides of them [for example peptide fusions LB1 (f); US 5843464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortees)]; P6 [EP 281673 (State University of New York)]; TbpA and / or TbpB; Hia; Hsf; Hin47; Hif; Hmw1; Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); P2; and P5 (WO 94/26304). The proteins of the invention can also be combined beneficially. By "combination" it is meant that the immunogenic composition contains all the proleins of the following combinations, either as proleins or as free proleins or as a mixture of the two. For example, in a combination of two proteins that is outlined here further, both proleins can be used as carrier proleins, or both proteins can be present as free proteins, or both can be present as a porler and as a free protein, or one can be present as a carrier prolein and a free protein while the olra is present only as a booster protein or only as a free protein, or one can serve as a carrier protein and the other as a free protein. When a combination of three proteins is given, there are similar possibilities. Preferred combinations include, but are not limited to, chimeric PhtD + NR1xR2, PhtD + NR1xR2-Sp91 at term C or fusion proteins, PhtD + PIy, PhtD + Sp128, PhtD + PsaA, PhtD + PspA, PhtA + NR1xR2, PhtA + NR1xR2-Sp91 chimeric in the C terminus or fusion proteins, PhtA + PIy, PhtA + Sp128, PhtA + PsaA, PhtA + PspA, NR1xR2 + LytC, NR1xR2 + PspA, NR1xR2 + PsaA, NR1xR2 + Sp128, R1xR2 + LytC, R1xR2 + PspA, R1xR2 + PsaA, R1xR2 + Sp128, R1xR2 + PhtD, R1xR2 + PhtA. Preferably, NR1xR2 (or R1xR2) is from CbpA or PspC. More preferably this is from CbpA. Other combinations include combinations of 3 proteins such as PhtD + N R1 xR2 + PIy, and PhtA + N R1 xR2 + PhtD. In one embodiment, the vaccine composition contains detoxified pneumolysis and PhtD or PhtDE as porous proteins. In a further embodiment, the vaccine composition contains detoxified pneumolysin and PhtD or PhtDE as free proiein. In an independent aspect, the present invention provides an immunogenic composition which contains at least four capsular saccharide conjugates of S. pneumoniae containing saccharides of different serotypes of S. pneumoniae, wherein at least one saccharide is conjugated to PhtD or prolein of fusion of them and the immunogenic composition is capable of triggering an effective immune response against PhtD. An effective immune response with PhlD or fusion proleins thereof is measured for example by means of a protection analysis, such as those described in Example 1 5. An effective immune response provides at least 40%, 50%, 60% , 70%, 80% or 90% survival 7 days after exposure with a heterologous strain. Since the exposure strain is heterologous, the protection achieved is due to the immune response against PhtD or its fusion prolein. Allernatively, an effective immune response with PhiD is measured by EL I SA as described in example 1 4. An effective immune response produces an anti-PhtD IgG response of at least 250, 300, 350, 400, 500, 550 or 600 μg / m LG MC.

For example, the immunogenic composition contains at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 capsular saccharides of S. pneumoniae of different serotypes conjugated with PhtD or fusion protein thereof. For example serotypes 22F and 1, 2, 3, 4, 5, 6 or 7 selected in addition to serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 23F and 33F are conjugated with PhtD. In a two or three modality serotypes 3, 6A and 22F are conjugated with PhtD or fusion proteins thereof.

In one embodiment, the immunogenic composition of the invention contains at least one capsular saccharide of S. pneumoniae conjugated to PhtD or fusion protein thereof by a linker, for example ADH. In one embodiment, one of the conjugation chemistries listed below is used. In one embodiment, the immunogenic composition of the invention contains at least one capsular saccharide of S. pneumoniae conjugated to PhtD or fusion protein thereof, wherein the ratio of PhtD to saccharide in the conjugate is between 6: 1 and 1. : 5, 6: 1 and 2: 1, 6: 1 and 2.5: 1, 6: 1 and 3 1, 6 1 and 35: 1 (w / w) or is greater than (that is, it contains a higher proportion of PhtD) 2 0: 1, 2.5-1, 3.0-1, 3.5: 1 or 4.0: 1 (w / w) In one embodiment, the immunogenic composition of the invention contains pneumolysin. The present invention further provides a vaccine containing the immunogenic compositions of the invention and a pharmaceutically acceptable excipient.

The vaccines of the present invention can be aided with an adjuvant, particularly when they are intended for use in a population of elderly adults, but also for use in infant populations. The adjuvants include an aluminum salt, such as aluminum oxide hydroxide or alum or aluminum phosphate, but it can also be other metal salts, such as calcium, magnesium, iron or zinc, or it can be an insoluble suspension. of acylated tyrosine, or acylated sugars, anionically or cationically derived saccharides, or polyphosphazenes. It is preferred that the adjuvant be selected to be a preferential inducer of a TH 1 type response. These high levels of Th 1 type cytokines tend to favor the induction of immune responses mediated by cells for a given antigen, while high levels of Th2 type cytokines tend to favor the induction of humoral immune responses to the antigen. The distinction of the Th 1 and Th 2 type immune response is not absolute. In fact, an individual will maintain an immune response that is described as predominantly Th 1 or predominantly Th 2. However, it is often convenient to consider the families of cytokines in terms of what was described in Murine CD4 + ve T cell clones by Mosmann and Coffman (Mosmann, T. R. and Coffman, R. L. (1989); TH 1 and TH2 cel ls: different patterns of lymphokine secretion lead to different functional properties. (Annual Review of Immunology, 7, p 1 45- 1 73). Traditionally, Th1 type responses are associated with the production of INF-? cytokines. and I L-2 by T lymphocytes. Other cytokines are frequently directly associated with the induction of Th1-type immune responses not produced by T cells, such as IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6, I L-10. Suitable adjuvant systems that promote a predominantly Th1 response include: Monosaccharide lipid A or a derivative thereof (or detoxified lipid A in general - see for example WO2005107798), particularly lipid A 3-de-O-acylated monophosphoryl (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 example aluminum phosphate or aluminum hydroxide) or an oil-in-water emulsion. In these combinations, the antigen and the 3D-MPL are contained in the same particle structures, making possible a more efficient supply of antigen and immunostimulatory signals. Studies have shown that 3D-MPL is able to further improve the immunogenicity of an antigen adsorbed on alum [Thoelen et al. Vaccine (1998) 16: 708-14; EP 689454-B1]. An improved system involves the combination of a lipid A monophosphoryl and a saponin derivative, particularly the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition wherein the QS21 is extinguished with cholesterol as described in WO 96/33739. A particularly potent adjuvant formulation involving QS21, 3D-M PL and tocopherol in an oil-in-water emulsion is described in WO 95/1 721 0. In one embodiment, the immunogenic composition additionally contains a saponin, which may be QS21. The formulation may also include an oil in water emulsion and tocopherol (WO 95/1721 0). Non-methylated CpG containing oligonucleotides (WO 96/02555) and other immunomodulatory oligonucleotides (WO0226757 and WO03507822) are also preferential inducers of a TH 1 response and are suitable for use in the present invention. Particular adjuvants are those selected from the group of metal salts, oil-in-water emulsions, similar receptors, a toll-like receptor agonist, (in particular, toll-type receptor 2 agonist, t-receptor-3 receptor agonist, receptor agonist). 4 of toll-type, agonist of toll-like receiver 7, agonist of toll-like receiver 8 and agonist of receiver 9 of t tol toll), saponines or combi nations of them. An adjuvant that can be used with the vaccine compositions of the invention are blisters or outer membrane vesicle preparations of Gram-negative bacterial strains, such as those presented in WO02 / 09746 - particularly N-ampoules. meningitidis. The adjuvant properties of the ampoules can be improved by keeping LOD (lipooligosaccharide) on its surface (for example by extraction at concentrations low detergent [eg 0-0.1% deoxycholate]). LOS can be detoxified through mutations msbB (-) or htrB (-) described in WO02 / 09746. The properties of the adjuvant can also be improved by preserving PorB (and optionally eliminating PorA) from the meningococcal bullae. The properties of the adjuvant can also be improved by truncating the saccharide structure exterior to the core of the meningococcal bullae - for example, by the IgtB (-) mutation described in WO2004 / 01441 7. Alternatively, the aforementioned LOS (eg isolates of a msbB strain (-) and / or IgtB (-)) can be purified and used as an adjuvant in the compositions of the invention. Another adjuvant that can be used with the compositions of the invention can be selected from the group of a saponin, lipid A or a derivative thereof, an immunostimulatory oligonucleotide., an alkyl glucosaminide phosphate, an oil in water emulsion or combinations of them. An additionally preferred adjuvant is a metal salt in combination with another adjuvant. It is preferred that the adjuvant be a toll-type receptor agonist, in particular an agonist of a 2, 3, 4, 7, 8 or 9 receptor of the toll or a saponin, in particular Qs21. It is further preferred that the adjuvant system contains two or more adjuvants from the above list. In particular, the combinations preferably contain a saponin (in particular Qs21) adjuvant and / or a toll-like receptor 9 agonist, such as a CpG containing immunostimulatory oligonucleotide. Other preferred combinations comprise a saponin (in particular QS21) and a toll-like receptor 4 agonist, such as lipid A monophosphoryl or its deacylated derivative 3 D-MPL, or a saponin (in particular QS21) and a toll-like receptor 4 ligand, such as an alkyl glucosamide phosphate. Particularly preferred adjuvants are combinations of 3D-MPL and QS21 (EP 0 671 948 B1), oil-in-water emulsions containing 3D-MPL and QS21 (WO 95/17210, WO 98/56414), or 3D-MPL formulated with other carriers (EP O 689 454 B1). Other preferred adjuvant systems include a combination of 3 D MPL, QS21 and a CpG oligonucleotide as described in US6558670, US6544518. In one embodiment, the adjuvant is (or contains) a toll-like receptor 4 (TLR) ligand, preferably an agonist, such as a lipid A derivative, particularly lipid A monophosphoryl or more particularly deacylated monophosphoryl lipid 3 (3 D) - MPL). 3D-MPL is available from GlaxoSmithKine Biologicals North America and primarily promotes CD4 + T cell responses with an IFN-g (Th1) phenotype. This can be produced according to the methods described in GB 2 220 211 A. Chemically, this 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 the small particle 3 D-MPL is used. The small particle 3 D-MPL has such a particle size that it can be filtered sterile through a 0.22 μm filter. These preparations are described in the application International Patent No. WO 94/21292. The synthetic derivatives of Lipid A are known and are thought to be TLR 4 agonists, including, but not limited to: OM 174 (2-deoxy-6-o- [2-deoxy-2 - [(R) -3- dodecanoyloxytetra-decanoi lamino] -4-o-phosphono-β-Dg I-ucopyranosyl] -2 - [(R) -3-hyd rox and tetra-decanoylamino] -aD-glucopyranosyl dihydrogenphosphate), (WO 95/14026) OM 294 DP (3S, 9R) -3 - [(R) -dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 (R) - [(R) -3-hydroxytetradecanoylamino] decan-1, 10-diol, 1, 10-bis (dihydrogenphosphate) (WO99 / 64301 and WO 00/0462) OM 197 MP-Ac DP (3S-, 9R) -3 - [(R) -dodecanoyloxy-tetradecanoylaminoH-oxo-S-aza-9 - [( R) -3-hydroxytetradecanoylamino] decan-1, 10-diol, 1-dihydrogenphosphate 10- (6-aminohexanoate) (WO 01/46127) Other TLR4 ligands that can be used are alkyl glucosaminide phosphates (AGP) such as those described in WO9850399 or US6303347 (also described processes for the preparation of AGP), or pharmaceutically acceptable salts of AGP, as described in US6764840. Some AGPs are TLR4 agonists and some are TLR4 antagonists. It is thought that both are useful as adjuvants. Another preferred 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 Saponaria Molina and was first described with adjuvant activity by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254). Purified fragments of Quil A have been isolated by HPLC, which maintain 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 derived from the bark of Quillaja saponaria Molina that induces CD8 + cytotoxic T cells (CTLs), TM 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, these formulations further comprise a sterol (WO96 / 33739). The saponins which form part of the present invention can be separated in the form of micelles, mixed micelles (preferably, but not exclusively, with bile salts) or can have the form of ISCOM matrices (EP 0 109 942 B1), liposomes or related colloidal structures, such as worm-type or ring-type multimeric complexes or layered and lamellar lipid structures when formulated with cholesterol and lipid, or in the form of an oil in water emulsion (for example in WO 95/17210). Saponins may preferably be associated with a metal salt, such as aluminum hydroxide or aluminum phosphate (WO 98/15287). Preferably, the saponin is presented in the form of a liposome, ISCOM or an oil-in-water emulsion. An improved system involves the combination of a lipid A monophosphoryl (oligoid A detoxified) and a saponin derivative, particularly the combination of QS21 and 3D-M PL as described in WO 94/001 53, or a less reactogenic composition wherein QS21 is quenched with cholesterol as described in WO 96/33739. A particularly potent adjuvant formulation involving tocopherol with or without QS21 and / or 3D-M PL in an oil-in-water emulsion is described in WO 95/1 721 0. In one embodiment the additional uninogenic composition contains a saponin, which It can be QS21. The immunostimulatory oligonucleotides or any other toll-like receptor (TLR) agonist 9 can be used. The preferred oligonucleotides for use in adjuvants or vaccines of the present invention are CpG containing oligonucleotides, preferably containing two or more CpG dinucleotide motifs separated by at least three, more preferably at least six or more nucleotides. A CpG motif is a nucleotide Cytosine followed by a Guanine nucleotide. The CpG oligonucleotides of the present invention are commonly deoxynucleotides. In a preferred embodiment, the internucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate linkage, although phosphodiester linkages and other internucleotide linkages are within the scope of the invention. Oligonucleotides with mixed internucleotide linkages are also included within the scope of the invention. Methods for producing phosphorothioate or phosphorodithioate oligonucleotides are described in US Pat. No. 5,666,153, US Pat. No. 5,278,302 and WO95 / 26204.

Examples of preferred oligonucleotides have the following sequences. The sequences preferably contain modified phosphorothioate internucleotide linkages. OLIGO 1 (SEQ ID NO: 1): TCC ATG ACG TTC CTG ACG TT (CpG 1 826) OLI GO 2 (SEQ ID NO: 2): TCT CCC AGC GTG CGC CAT (CpG 1 758) OLIGO 3 (S EQ ID NO: 3): ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG OLI GO 4 (SEQ ID NO: 4): TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) OL I GO 5 (SEQ ID N0: 5): TCC ATG ACG TTC CTG ATG CT (CpG 1 668) OLIGO 6 (SEQ ID N0: 6): TCG ACG TTT TCG GCG CGC GCC G (CpG 5456 The CpG oligonucleotides can contain the above preferred sequences, in which there are deletions or inconsistent additions thereto. The CpG oligonucleotides used in the present invention can be synthesized by any method known in the art (for example see EP 468520). Conveniently, these oligonucleotides can be synthesized using an automated synthesizer. The adjuvant may be an oil-in-water emulsion or may contain an oil-in-water emulsion in combination with other adjuvants The oil phase of the emulsion system preferably contains a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "capable of being transformed by metabolism" (Dorland's Illustrated Medical Dictionary, W. B. Sanders Company, 25th edition (1974)). The oil can be any vegetable, fish, animal or synthetic oil, which is not toxic to the recipient, and which can be transformed by the metabolism. Nuts, seeds and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and may include commercially available oils, such as NEOBEE® and others. Squalene (2,6,10,15,19, 23-Hexamethyl-2,6,10,14,18,22-tetracosahexaeno) is an unsaturated oil found in large quantities in shark liver oil, and in Smaller amounts are found in olive oil, wheat germ oil, rice bran oil, and yeast, and it is a particularly preferred oil for use in this invention. Squalene is a metabolizable oil by virtue of the fact that it is an intermediate product in cholesterol biosynthesis (Merck index, 10th edition, entry no.8619). Touches (for example, vitamin E) are frequently used in adjuvant emulsions (EP 0 382 271 B1; US5667784; WO 95/17210). The knobs used in the oil emulsions (preferably oil-in-water emulsions) of the invention can be formulated as described in EP 0 382 271 B1, in which the knobs may be dispersions of tocol droplets, optionally with an emulsifier, preferably less than 1 m in diameter. Alternatively, the knobs may be used in combination with another oil, to form the oily phase of an oil emulsion. Examples of oil emulsions that can be used in combination with tocol are described herein, such as the metabolizable oils described above. It has been suggested that oil-in-water emulsion adjuvants per se are useful as adjuvant compositions (EP 0 399 843B), also combination of oil-in-water emulsions and other active agents have been described as adjuvants for vaccines (WO 95 / 1 721 0, WO 98/5641 4, WO 99/1 2565, WO 99/1 1 241). Other emulsion adjuvants in oil have been described, such as oil-in-oil emulsions (US 5, 422, 1 09; 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 (particularly when touches are incorporated) to form adjuvants and compositions of the present invention. Most preferably the oil emulsion (for example oil-in-water emulsions) further contains an emulsifier such as TWE EN 80 and / or a sterol such as cholesterol. A preferred oil emulsion (preferably oil in water emulsion) contains a metabolizable, non-toxic oil, such as squalane, squalene or a tocopherol, such as alpha tocopherol (and preferably both squalene and alpha tocopherol) and optionally a emulsifier (or surfactant) such as Tween 80. A sterol (preferably cholesterol) may also be included. The method for producing oil-in-water emulsions is well known to those skilled in the art. Commonly, the method comprises mixing the oil phase containing tocol with a surfactant such as a solution of PBS / TWEEN80 ™ solution, followed by homogenization using a homogenizer, it may be clear to a person skilled in the art that a method comprising passing the mixture twice through a needle may be appropriate to homogenize small volumes of liquid. Likewise, the process of emulsification in a microfluidizing machine (M 1 1 0S M icrofl uidics) maximum of 50 steps, during a period of 2 min utes a maximum pressure input of 50 steps, during a period of 2 minutes to a maximum inlet pressure of 600 kPa (6 bar) (outlet pressure of about 8500 kPa (850 bar))) could be adapted by the person trained in the art to produce smaller or larger emulsion volumes. The adaptation could be achieved by routine experimentation comprising the measurement of the resulting emulsion until a preparation with oil droplets of the required diameter is obtained. In an oil-in-water emulsion, the oil and the emulsifier must be in an aqueous carrier. The aqueous carrier can be, for example, phosphate-regulated salt. The size of the oil droplets found within the The stable oil-in-water emulsion is preferably less than 1 miera, it may be in the range of substantially 30-600 nm, preferably substantially around 30-500 nm in diameter, and much more preferably substantially 1 50-500 nm in diameter, and in particular approximately 1 50 nm in diameter as measured by photon correlation spectroscopy. In this regard, 80% of the oil droplets per number should be within the preferred ranges, more preferably more than 90% and much more preferably more than 95% of the oil droplets per amount are within the defi size ranges. Nest The amounts of the components present in the oil emulsion of the present invention are conventionally in the range from 0 5-20% or 2 to 10% oil (of the total dose volume), such as squalene, and when present, from 2 to 10% alpha tocopherol, and from 0.3 to 3% surfactant, such as polyoxyethylene sorbitan mono oleate. Preferably the ratio of oil (preferably squalene) tocol (preferably α-tocopherol) is equal to or less than 1 since it provides a more stable emulsion. An emulsifier, such as Tween 80 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 invention will also contain a stabilizer. Examples of preferred emulsion systems are described in WO 95/1 721 0, WO 99/1 1 241 and WO 99/1 2565, which describe emulsion adjuvants based on squalene, α-tocopherol, and TWEEN 80, optionally formulated with the immunostimulants QS21 and / or 3D-M PL. Thus, in a particularly preferred embodiment of the present invention, the adjuvant of the invention may additionally contain other immunostimulants, such as LPS or its derivatives, and / or saponins. Examples of other immunostimulants are described here and in Vaccine Design - The subunit and adyuvant approach "1 995 Pharmaceutical Biotechnology, Volume 6, Eds Powell, MF, and Newman, MJ., Plenum Press, New York and London, ISB N 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-M PL) in an oil emulsion which is described above, optionally with a sterol (preferably cholesterol) Additionally, the oil emulsion (preferably oil-in-water emulsion) may contain span 85 and / or lecithin and / or tricapryly.Adjuvants containing an oil-in-water emulsion , a sterol and a saponin are described in WO 99/1 2565. Commonly for administration to humans saponin (preferably QS21) and / or LPS derivative (preferably 3D-M PL) will be present in a human dose. of immunogenic composition in the range of 1 μg - 200 μg, such as 1 0-1 00 μg, preferably 1 0 μg - 50 μg per dose. Commonly, the oil emulsion (preferably oil-in-water emulsion) will contain from 2 to 10% of metabolizable oil.

Preferably it will contain from 2 to 10% of squalene, from 2 to 10% of alpha tocopherol and from 0.3 to 3% (preferably 0.4-2%) of emulsifier (preferably tween 80 [polyoxyethylene sorbitan mono oleate]). Where squalene and alpha tocopherol are present, preferably the squalene alpha tocopherol ratio is less than or equal to 1, since this provides a more stable emulsion. Span 85 (Sorbitan trioleate) may also be present at a level from 0.5 to 1% in the emulsions used in the invention. In some cases it may be advantageous if the immunogenic compositions and vaccines of the present invention additionally contain a stabilizer, for example other emulsifiers / surfactants, including caprylic acid (Merck index 1st edition, entry No. 1 739), of which Tricaprylin is particularly preferred. When squalene and a saponin are included (preferably QS21), it is beneficial to also include a sterol (preferably cholesterol) in the formulation, since this allows a reduction in the total oil level in the emulsion. This leads to a reductive manufacturing cost, improvement of the total comfort of the vaccination, and also quantitative and qualitative improvements of the resulting immune responses, such as improved production of I FN- ?. Accordingly, the adjuvant system of the present invention commonly contains a metabolizable ratio of oil: saponin (w / w) in the range of 200: 1 to 300: 1, also the present invention can be used in a "low in oil" whose preferred range is 1.1 to 200.1, preferably 20.1 to 100.1, and much more preferably substantially 48.1, this vaccine maintains the beneficial adjuvant properties of all components, with a very reduced reactogenicity profile. Accordingly, the particularly preferred embodiments have a squalene ratio QS21 (w / w) in the range from 11 to 250.1, a range from 20.1 to 200.1, preferably 20.1 to 100.1, and much more preferably substantially 48.1 is also preferred. a sterol (much more preferably cholesterol) also included, has a ratio of saponin sterol as described herein. The emulsion systems of the present invention preferably have a small droplet size in the sub-micron range. Much more preferably the sizes of oil droplets will be in the range from 120 to 750 nm, and much more preferably from 120 to 600 nm in diameter. A particularly potent adjuvant formulation (for final combination with AIP04 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 a emulsion of oil in water) as described in WO 95/17210 or in WO 99/12565 (in particular, the 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), while double-stranded RNA and poly IC (polyinosin-potidyl acid - a synthetic commercial imitation of viral RNA), are examples of TLR 3. The 3D-MPL is an example of a TLR4 agonist, while the CPG is an example of a TLR9 agonist. The immunogenic composition may contain an antigen and an immunostimulant adsorbed on a metal salt. Aluminum-based vaccine formulations wherein the antigen and the 3-de-O-acylated monophosphoryl lipid A immuno-stimulant (3D-MPL) are adsorbed on the same particle are described in EP 0 576478 B1, EP 0689454 B1, and EP 0633784 B1. In these cases the antigen is adsorbed first on the aluminum salt followed by the adsorption of the 3D-MPL immunostimulant on the same aluminum salt particles. These processes first involve the suspension of 3D-MPL by sound treatment in a water bath until the particles reach a size between 80 and 500 nm. The antigen is commonly adsorbed on aluminum 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 maintained at 4 ° C until use. In another process, the immunostimulant and the antigen are in separate metallic particles, as described in EP 1 1 26876. The improved process contains the adsorption of immunostimulant, in a metal salt particle, followed by the adsorption of the antigen on another metal salt particle, followed by the mixing of the particles discrete metal to form a vaccine. The adjuvant for use in the present invention may be an adjuvant composition containing an immunostimulant, adsorbed on a metal salt particle, characterized in that the metal salt particle is substantially free of another antigen. Additionally, vaccines are provided by the present invention, and are characterized in that the immunostimulant is adsorbed on metal salt particles that are substantially free of another antigen, and because the metal salt particles that are adsorbed on the antigen are substantially free of other immunostimulant. Accordingly, the present invention provides an adjuvant formulation containing an immunostimulant which has been adsorbed on a metal salt particle, which is characterized in that the composition is substantially free of another antigen. Moreover, this adjuvant formulation can be an intermediate product which, if this adjuvant is used, is required for the manufacture of a vaccine. Accordingly, there is provided a process for the manufacture of a vaccine comprising mixing an adjuvant composition consisting of one or more immunostimulants adsorbed on a metal particle with a antigen Preferably, the antigen has been pre-adsorbed on a metal salt. Said metal salt may be identical or similar to the metal salt which is adsorbed on the immunostimulant. Preferably the metal salt is an aluminum salt, for example aluminum phosphate or aluminum hydroxide. The present invention also provides a vaccine composition containing immunostimulant adsorbed on a first particle of a metal salt, and antigen adsorbed on a metal salt, characterized in that the first and second metal salt particles are separate particles. The derivatives or mutations of LPS or LOS or lipid derivative A described herein are designed to be less toxic (eg 3D-M PL) than the natural lipopolysaccharides, and are interchangeable equivalents with respect to any uses of these portions described here. They can be TLR4 ligands as described in the above. Other of these derivatives are described in WO020786737, WO9850399, WO01 3461 7, WO021 2258, WO03065806. In one embodiment, the adjuvant that is used for the compositions of the invention contains a liposome carrier (made by known techniques from a phospholipid (such as dioleoyl phosphatidyl choline [DOPC]) and optionally a sterol [such as cholesterol]. ]). These liposome carriers can carry lipid A derivatives [such as 3D-M PL - see above] and / or saponins (such as QS21 - see above). In one modality, the adjuvant contains (per dose of 0.5 mL) 0.1-10mg, 0.2-7, 0.3-5, 0.4-2, or 0.5-1 mg (for example 0.4-0.6, 0.9-1.1, 0.5 or 1 mg) of phospholipid (per DOPC example), 0.025-2.5, 0.05-1.5, 0.075-0.75, 0.1-0.3, or 0.125-0.25 mg (for example 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg) of sterol (for example cholesterol), -60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of lipid derivative A (e.g. 3D-MPL), and 5- 60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) saponin (for example QS21). This adjuvant is particularly suitable for vaccine formulations for the elderly. In one embodiment the vaccine composition containing this adjuvant contains saccharide conjugates originating from at least all of the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also include one or more of serotypes 3, 6A, 19A, and 22F), where the GMC antibody titer induced against one or more (or all) components of vaccine 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates. In one embodiment, the adjuvant used for the compositions of the invention contains an oil-in-water emulsion made from a metabolizable oil (such as squalene), an emulsifier (such as Tween 80) and optionally to tocol (such as alpha tocopherol). ). In one embodiment, the adjuvant contains (per dose of 0.5 mL) 0.5-15, 1-13, 2-1 1, 4-8, or 5-6mg (for example 2-3, 5-6, or 10-11 mg) metabolizable oil (such as squalene), 0.1-10, 0.3-8, 0. 6-6, 0.9-5, 1-4, or 2-3 mg (for example 0.9-1.1, 2-3 or 4-5 mg) of emulsifier (such as Tween 80) and optionally 0.5-20, 1-15 , 2-12, 4-10, 5-7 mg (for example 11-13, 5-6, or 2-3 mg) of tocol (such as alpha tocopherol). This adjuvant optionally can also contain 5-60, -50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of lipid A derivative (e.g. 3D-MPL). These adjuvants are particularly suitable for vaccine formulations for infants or for the elderly. In one embodiment the vaccine composition containing this adjuvant contains saccharide conjugates originating from at least all of the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also include one or more of serotypes 3, 6A, 19A, and 22F), where the GMC antibody titer induced against one or more (or all) components of vaccine 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates. 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 (eg 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg) of sterol (eg cholesterol), 5-60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of lipid derivative A (e.g. 3D-MPL), and -60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of saponin (for example QS21). This adjuvant is particularly appropriate for vaccine formulations for the elderly. In one embodiment the vaccine composition containing this adjuvant contains saccharide conjugates derived from at least all of the following serotypes: 4, 6B, 9V, 14, 18d 19Ft 23F, 1, 5, 7F (and may also include one or more of the serotypes 3, 6A, 19A, and 22F), where the GMC antibody titer induced against one or more (or all) vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than induced by the Prevnar® vaccine in vaccinated humans. In one embodiment the adjuvant used for the compositions of the invention contains aluminum phosphate and a lipid derivative A (such as 3D-MPL). This adjuvant may contain (per dose of 5 mL) 100-750, 200-500, or 300-400 μg of Al as aluminum phosphate, and 5-60, 10-50, or 20-30 μg (for example 5- 15, 40-50, 10, 20, 30, 40 or 50 μg) of lipid derivative A (e.g. 3D-MPL). This adjuvant is particularly suitable for infant vaccine formulations or for elderly adults. In one embodiment, the vaccine composition containing this adjuvant contains saccharide conjugates from at least all of the following serotypes:, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also include one or more of serotypes 3, 6A, 19A, and 22F), wherein the GMC antibody titer induced against one or plus (or all) vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F are not significantly lower than those induced by the vaccine Prevnar® in vaccinated humans. The vaccine preparations containing immunogenic compositions of the present invention can be used to protect or treat a susceptible mammal by administering said vaccine systemically or mucosally. These administrations may include injection by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or by mucosal administration to the oral / ali mentary, respiratory, genitourinary tract. The intranasal administration of vaccines for the treatment of pneumonia or otitis media is preferred (since the nasopharyngeal carriage of pneumococci can be prevented more effectively, thereby attenuating infection at this early stage). Although the vaccine of the invention can be administered as a single dose, its components can also be co-administered together at the same time or at different times (for example the pneumococcal saccharide conjugates could be administered separately, at the same time or at the same time). -2 weeks after the administration of any component of the bacterial protein of the vaccine for optimal coordination of the immune responses with respect to the rest of them). For co-administration, the optional Th 1 adjuvant may be present in any or all of the different administrations. In addition to a single administration path, 2 different administration routes can be used. For example, saccharides or saccharide conjugates can be administered I M (or I D) and bacterial proteins can be administer IN (or ID). In addition, the vaccines of the invention can be administered IM for sensitization doses and IN for booster doses. The content of protein antigens in the vaccine will commonly be in the range of 1-100 μg, preferably 5-50 μg, much more commonly in the range of 5-25 μg. After an initial vaccination, the subjects may receive one or several booster immunizations properly separated. The vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell M F. and Newman M.J.) (1995) Plenum Press New York). Encapsulation with liposomes is described in Fullerton, US Patent 4,235,877. The vaccines of the present invention can 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 preferable that they are lyophilized and reconstituted extemporaneously before use. Lyophilization 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. In one aspect of the invention, a vaccine kit is provided, which includes a bottle containing an immunogenic composition of the invention, optionally in lyophilized form, and which also includes a bottle containing an adjuvant as described herein. It is anticipated that in this aspect of the invention, the adjuvant will be used to reconstitute the lyophilized immunogenic composition. While the vaccines of the present invention can be administered by any route, administration of the described skin (ID) vaccines forms one embodiment of the present invention. Human skin contains a "callous" outer cuticle called stratum corneum, which rests on the epidermis. Beneath this epidermis is a layer called the dermis, which in turn rests on the subcutaneous tissue. Researchers have shown that injection of a vaccine into the skin, and in particular into the dermis, stimulates an immune response, which may also be associated with a number of additional benefits. Intradermal vaccination with the vaccines described herein forms a preferred feature of the present invention. The conventional technique of intradermal injection, the "Mantoux procedure" includes the steps of cleaning the skin, and then stretch it with one hand, and with the bezel of a needle of thin gauge (caliber 26-31) oriented upwards the needle is inserted at an angle of between 10-15 °. Once the bevel of the needle is inserted, the plunger of the needle is lowered and advanced by providing a slight pressure to raise it to the skin. Then the liquid is injected very slowly, forming a blister or bump on the surface of the skin, followed by removal slow of the needle. More recently, devices that are specifically designed to deliver liquid agents in or through 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 5,366,120, 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 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 the ballistic delivery of solid vaccines (WO 99/27961), or transdermal patches (WO 97/48440, WO 98/28037); or application to the surface of the skin (transdermal or transcutaneous administration WO 98/20734, WO 98/28037). When the vaccines of the present invention are to be administered to the skin, or more specifically to the dermis, the vaccine has a low volume of liquid, particularly a volume of between about 0.05 ml and 0.2 ml. The content of skin antigens or intradermal vaccines of the present invention may be similar to the conventional doses found in intramuscular vaccines. (see above). However, a characteristic of skin vaccines or intradermal vaccines that the formulations can be "low dose". Accordingly, protein antigens in "low dose" vaccines are preferably present in as little as 0. 1 to 10 μg, preferably 0.1 to 5 μg per dose; and the saccharide antigens (preferably conjugated) may be present in the range of 0.01 -1 μg, and preferably between 0.01 to 0.5 μg of saccharide per dose. As used herein, the term "intradermal delivery" means the delivery of the vaccine to the dermis region in the skin. However, the vaccine will not necessarily be located exclusively in the dermis. The dermis is the layer on the skin located between about 1.0 and about 2.0 mm from the surface on human skin, but there is a certain amount of variation between individuals and in different parts of the body. In general, you can expect 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 on the surface and in the subcutaneous layer below. Depending on the form of administration, the vaccine can finally be located only or primarily within the dermis, or it can finally be distributed within the epidermis and dermis. The present invention further provides an improved vaccine for the prevention or amelioration of otitis media produced by Haemophilus influenzae by the addition of proteins of Haemophilus influenzae, for example protein D in free or conjugated form. In addition, the present invention also provides an improved vaccine for the prevention or amelioration of pneumococcal infection in infants (e.g., otitis media), supported upon the addition of one or two pneumococcal proteins as free or conjugated protein to conjugate compositions of S. pneumoniae of the invention. Said pneumococcal free proteins may be the same as or different from any S. pneumoniae proteins used as carrier proteins. U n or more Moraxella catarrhalis protein antigens can also be included in the combination vaccine in a conjugated and conjugated form. Thus, the present invention is an improved method for eliciting an immunitary (protective) response against otitis media in infants. In another embodiment, the present invention is an improved method for eliciting an immune (protective) response in infants (defined as 0-2 years of age in the context of the present invention) by administering a safe and effective amount of the vaccine. of the invention [a pediatric vaccine]. Other embodiments of the present invention include the provision of the antigenic compositions of S. pneumoniae conjugates of the invention for use in medicine and the use of the conjugates S. pneumoniae of the invention in the manufacture of a medicament for the prevention ( or treatment) of pneumococcal disease In yet another modality, the present invention is a method improved to elicit an immune (protective) response in the elderly population (in the context of the present invention a patient is considered to be of advanced age if he is 50 years of age or older, commonly more than 55 years and more generally more than 60 years) administering a safe and effective amount of the vaccine of the invention, preferably in conjunction with one or two proteins of S. pneumoniae present as free or conjugated protein, whose free S. pneumoniae proteins can be the same or different from any S. pneumoniae proteins used as carrier proteins. A further aspect of the invention is a method for immunizing a human host against disease caused by S. pneumoniae and optionally Haemophilus influenzae infection which comprises administering to the host an immunoprotective dose of the immunogenic composition or vaccine or equipment 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 infection by Haemophilus influenzae. A further aspect of the invention is the 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 infection by Haemophilus influenzae The terms "containing", "comprises" and "contains" here they intend on the part of the inventors to be optionally substitutable with the terms "consisting of," consisting of and "consists, respectively, in each case The embodiments presented herein called" vaccine compositions "of the invention are also applicable to modalities termed" immunogenic compositions "of the invention, and vice versa.All references or patent applications cited within This patent specification is incorporated herein by reference.In order that this invention may be better understood, the following examples are set forth.These examples are for illustration purposes only, and should not be construed as limiting the scope of the invention. the invention in no way Examples Example 1: Protein D expression Protein D from Haemophilus influenzae Genetic construction for protein D expression i Nicial materials DNA that encodes protein D Protein D is highly conserved among H. influenzae of all serotypes and unclassifiable strains.The vector pH I C348 that contains the sequence of DNA encoding the entire protein gene has been obtained by Dr. A. Forsberg, Department of Medical Microbiology, Lund University, General Hospital Malmo, Malmo, Sweden. The DNA sequence of protein D has been published by Janson et al. (1991) Infect. Immun.59: 119-125. The expression vector pMG1 The expression vector pMG1 is a derivative of pBR322 (Gross et al., 1985) in which control elements derived from the bacteriophage were introduced? for the transcription and translation of foreign genes inserted (Shatzman et al., 1983). In addition, the Ampicillin resistance gene was exchanged with the Kanamycin resistance gene. The E. coli strain AR58 The E. coli strain AR58 was generated by transduction of N99 with an existence of P1 phage previously cultured in an SA500 derivative (galE :: TN10, lambdaKil cl857? H1). N99 and SA500 are K12 strains of E. coli obtained from Dr. Martin Rosenberg's laboratory at the National Institute of Health. The expression vector pMG 1 For the production of protein D, the DNA encoding the protein has been cloned into the pMG 1 expression vector. This plasmid uses Lambda phage DNA signals to direct the transcription and translation of foreign genes inserted. The vector contains the PL promoter, the OL operator and two sites of use (NutL and NutR) to alleviate the effects of transcriptional polarity when N protein is provided (Gross et al., 1985). The vectors containing the PL promoter are introduced into a lysogenic host of E. coli to stabilize the plasmid DNA. The host strains Lysogens contain Lambda phage DNA with incomplete replication integrated into the genome. (Shatzman et al., 1983). The chromosomal Lambda phage DNA directs the synthesis of the repressor protein cl, which binds to the OL repressor of the vector and prevents the binding of the RNA polymerase to the PL promoter and therefore the transcription of the inserted gene. The cl gene of expression strain AR58 contains a temperature-sensitive mutant, so that PL-directed transcription can be regulated by temperature change, i.e. an increase in the temperature of the culture inactivates the repressor and the synthesis is initiated of the foreign protein. This expression system makes possible the controlled synthesis of foreign proteins, especially those that can be toxic to the cell (Shimataka and Rosenberg, 1981). E. coli strain AR58 The lysogenic strain E. coli AR58 used for the production of the carrier protein D is a derivative of the N99 strain of standard NIH E. coli K12 (F "su" galK2, lacZ "thr") . It contains an incomplete lysogenic lambda phage (gaE :: TN10, lambdaKir cl857? H1) The Kil phenotype "prevents the deactivation of the macromolecule synthesis of the host.The cl857 mutation confers a temperature sensitive lesion to the cl repressor. H1 removes the right operon from the lambda phage and the hosts bio, uvr3, and chIA loci.The AR58 strain was generated by N99 transduction with a P1 phage existence previously cultured in an SA500 derivative (galE :: TN10, lambdaKil "cl857? H1). The introduction of the incomplete lysogen in N99 is selected with tetracycline by virtue of the presence of a TN10 transposon that encodes tetracycline resistance in the adjacent galE gene. Construction of vector pMGMDPPrD Vector pMG 1 containing the gene encoding the non-structural protein S1 of Influenza virus (pMGNSI) was used to construct pMGMDPPrD. The protein D gene was amplified by PCR from the vector pHIC348 (Janson et al., 1991 Infect. Immun 59: 1 19-125) with PCR sensitizers containing the Ncol and Xbal restriction sites at the 5 'ends and 3 ', respectively. The Ncol / Xbal fragment was then introduced into pMGNSI between Ncol and Xbal thus creating a fusion protein containing the 81 amino acids of the N-terminus of the NS1 protein protein, followed by the PD protein. This vector was labeled pMGNSI PrD. Based on the construction described above, the final construct was generated for the expression of protein D. A BamHI / BamHI fragment of pMGNSI PrD was removed. This DNA hydrolysis removes the NS1 coding region, except for the first three residues at the N-terminus. With the religation of the vector, a gene encoding a fusion protein was generated with the following amino acid sequence at the N-terminus: MDP SSHSSNMANT NS1 Protein D Protein D does not contain a leader peptide or the N-terminal cysteine to which the lipid chains are normally bound.

Therefore neither the protein is excreted in the periplasm nor is it lipidated, and it remains in the cytoplasm in a soluble form. The final construct pMG-M DPPrD was introduced into host strain AR58 by heat shock at 37 ° C. Plasmids containing bacteria were selected in the presence of Kanamici. The presence of the DNA insert encoding protein D was demonstrated by digestion of isolated plasmid DNA with selected endonucleases. The recombinant strain of E. coli is called ECD4. Protein D expression is under the control of the lambda P / Operator 0L promoter. Host strain AR58 contains a cl gene sensitive to temperature in the genome, which blocks the expression of lambda PL at low temperature by binding to OL. Once the temperature is high, OL is released and protein D is expressed. Small scale preparation At the end of the fermentation, the cells are concentrated and frozen. The extraction of harvested cells and the purification of protein D was carried out as follows. The frozen cell culture granulate is thawed and resuspended in a solvent for interruption of cells (citrate buffer, pH 6.0) to a final OD650 = 60. The suspension is passed twice through a high pressure homogenizer. with P = 1 00000 kPa (1 000 bar). The cell culture homogenate is clarified by centrifugation and the cellular detritus is removed by filtration. In the first step of purification the filtered lysate is applied to a column for cationic exchange chromatography (SP Sepharose Fast Flow). The PD is fixed to the gel matrix by ionic interaction and eluted by a step of increasing the ionic strength of the buffer of the ion. In a second purification step, the impurities are retained in an anion exchange matrix (Q Sepharose Fast Flow). The PD is not fixed on the gel and can be collected in the flow that passes. In both steps of column chromatography, the collection of the fraction is monitored by OD. The flow through the anion exchange column chromatography containing the purified protein D is concentrated by ultrafiltration. Protein D containing the ultrafiltration retentate is finally passed through a 0.2 μm membrane. Large-scale preparation The extraction of harvested cells and the purification of protein D were carried out as follows. The culture broth is cooled and directly passed twice through a high pressure homogenizer at a pressure of about 80000 kPa (800 bar). In the first purification step the cell culture homogenate is diluted and applied to a column for cation exchange chromatography (SP Sepharose Big beads). The PD is fixed to the gel matrix by anionic interaction and is passed through a step of increasing the bionic resistance of the regulator of the solution and it is filtered. In a second purification step, the impurities are retained in an anion exchange matrix (Q Sepharose Fast Flow). The PD is not fixed on the gel and can be collected in the passing flow. In both steps of column chromatography, the collection of the fraction is monitored by OD. The flow through the anion exchange column chromatography containing the purified protein D is concentrated and subjected to diafiltration by ultrafiltration. Protein D containing the retentate from the ultrafiltration is finally passed through a 0.2 μm membrane. Example 1b: Expression of PhtD The PhtD protein is a member of the family of pneumococcal protein histidine in triad (Pht) characterized by the presence of histidine triads (motif HXXHXH). The PhtD is an 838 aa molecule and carries 5 triads of histidine (see Medl mm unites WO00 / 371 05 SEQ I D NO: 4 to examine the amino acid sequence and SEQ I D N O: 5 for the DNA sequence). PhtD also contains a region rich in proline in the medium (amino acid position 348-380). The PhtD has a signal sequence in the terminal N 20 aa with a LXXC motif. Genetic construction The genetic sequence of the mature protein PhtD Med l mmune (from aa 21 to a838) was recombinantly transferred to E. coli using the vector present pTCM P 1 4 promoter carrier p ?. The E. coli host strain is AR58, which carries the thermosensitive repressor cl857, which allows heat induction of the promoter. Polymerase chain reaction was performed to amplify the phtD gene of a Medlmmune plasmid (carrying the phtD gene of Streptococcus pneumoniae, strain Norway 4 (serotype 4) - SEQ ID NO: 5 as described in WO 00/37105). Sensitizers, specific for the phtD gene alone, were used to amplify the phtD gene in two fragments. The sensitizers carry any of the Ndel and Kpnl restriction sites or the Kpnl and Xbal sites. These sensitizers do not hybridize with any nucleotide in the vector, but only with genetic sequences specific for phtD. An artificial initiation codon ATG was inserted using the first sensitizer carrying the Ndel restriction site. The generated PCR products were then inserted into the cloning vector pGEM-T (Promega), and the DNA sequence was confirmed. Subcloning of the fragments into the TCMP14 expression vector was then performed using standard techniques and the vector transformed into AR58 from E coli. Purification of PhtD The purification of PhtD is achieved as follows: Culture of E coli cells in the presence of Kanamycin: culture for 30 hours at 30 ° C, then induction for 18 hours at 39.5 ° C. Rupture of whole-culture E coli cells in OD ± 115 in the presence of 5 mM EDTA and 2 mM PMSF as inhibitors of Protease: Rannie, 2 steps, 100000 kPa (1000 bar). Capture of antigen elimination of cell detritus in Streamline Q XL chromatography in pearl expanded at room temperature (20 ° C), the column is washed with 150 mM NaCl + Empigen 0.25%, pH 6.5 and eluted with 400 mM NaCl + Empigen 0. 25% in 25 mM potassium phosphate buffer with pH 7.4. Filtration in Sartobran 150 cartridge (0.45 + 0.2 μm) Fixation to antigen in IMAC FF chromatography with chelating sepharose in Zn ++ with a pH of 7.4 in the presence of 5 mM imidazole at 4 ° C, the column washed with 5 mM Imidazole and Empigen 1% and eluted with 50 mM imidazole, in regulator 25 mM potassium phosphate pH 8.0. Weak-effect anion chromatography in positive mode in Fractogel EMD DEAE with pH 8 0 (25 mM potassium phosphate) at 4 ° C, the column is washed with 140 mM NaCl and eluted in NaCl 200 mM while the contaminants (proteins and DNA) remain adsorbed in the exchanger. Concentration and ultrafiltration with Na 2 mM / K phosphate, with pH 7.15 in 50 kDa membrane. - Sterilization by filtration of the purified volume in a 0.2 μm Millipak-20 filter cartridge. Example 1c: Expression of pneumolysin Pneumococcal pneumolysin was prepared and detoxified as described in WO2004 / 081515 and WO2006 / 032499. Example 2: Preparing with ugados It is well known in the art how to make pneumococcal polysaccharides. For the purposes of these examples the polysaccharides were elaborated essentially as described in E P07251 3 or by closely related methods. Prior to conjugation the polysaccharides can be sized by microfluidization as described below. The activation and coupling conditions are specific for each polysaccharide. These are provided in Table 1. The sized polysaccharide (except for PS5, 6B and 23F) was dissolved in 2M NaCl, 0.2M NaCl or in water for injection (WFI). The optimal concentration of polysaccharide was evaluated for all serotypes. All serotypes except serotype 1 8C were conjugated directly to the carrier protein as detailed below. Two alternative serotypes were developed 22 F; one conjugate d i rightly, one by means of an ADH linker. Based on a mad solution of 1 00 mg / mL in acetonitriil or 50% / 50% acetonitrile / water solution, CDAP (ratio of CDAP / PS 0.5-1.5 mg / mg PS) was added to the polysaccharide solution. 1.5 minutes later, NaOH 0.2M-0.3M until obtaining the specific activation pH. Activation of the polysaccharide was carried out at this pH for 3 minutes at 25 ° C. The purified protein (protein D, PhtD, pneumolysin or DT) (the amount depends on the initial PS / carrier protein ratio) was added to the polysaccharide up to 2 hours (depending on the serotype) under pH regulation. In order to quench the cyanate ester groups, a 2M glycine solution was then added to the mixture. The pH was adjusted to the extinction pH (pH 9.0). The solution was stirred for 30 minutes at 25 ° C and then overnight for 2-8 ° C with slow continuous stirring. Preparation of 18C: 18C was linked to the carrier protein by a linker - Adipic acid dihydrazide (ADH). Serotype 18C polysaccharide was microfluidized before conjugation. Derivation of tetanus toxoid with EDAC For the derivation of tetanus toxoid, purified TT was diluted to 25 mg / mL in 0.2M NaCl and the ADH separator was added in order to reach a final concentration of 0.2M. When the dissolution of the separator was complete, the pH was adjusted to 6.2. Then EDAC (1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide) was added to a final concentration of 0.02M and the mixture was stirred for 1 hour under pH regulation. The condensation reaction was stopped by increasing the pH to 9.0 for at least 30 minutes at 25 ° C. The derivatized TT was then diafiltered (CO membrane, 10 kDa) in order to remove the residual ADH and the EDAC reagent. The mother TTAH was finally filtered to sterilize it and stored up to the coupling step at -70 ° C. Chemical coupling from TTAH to PS 18C The details of the conjugation parameters can be found in Table 1. 2 grams of microfluidized PS were diluted in the defined concentration in water and adjusted to 2M NaCl by the addition of NaCl powder. CDAP solution (100 mg / mL freshly prepared in 50/50 v / v acetonitrile / WFI) was added until reaching the appropriate CDAP / PS ratio. The pH was raised to the activation pH of 9.0 by the addition of 0.3M NaOH and stabilized at this pH until the addition of After 3 minutes, TTA H (20 mg / m L in 0.2 M NaCI) was added to reach a TTA H / PS ratio of 2; the pH was adjusted to the coupling pH of 9.0. The solution was left for one hour under pH regulation. For extinction, a glycine solution was added to the PS / TTA H / CDAP mixture. The pH was adjusted to the pH of extention (pH 9.0). The solution was stirred for 30 mm at 25 ° C and then left overnight for 2-8 ° C with slow continuous stirring. Conjugate PS22FAH-PhtD In a second conjugation method for this saccharide (the first being the conjugation method PS22-PhtD shown in Table 1), the 22F was linked to the carrier protein by a linker - Dihid acid adipic acid (ADH). He serotype 22F polysaccharide was microfluidized before conjugation. Derivation of PS 22F Activation and coupling were carried out at 25 ° C under continuous stirring in a water bath with controlled temperature. Microfluidized PS22F was diluted until a final PS concentration of 6 mg / mL in NaCl 0.2M was obtained and the solution was adjusted to a pH 6. 05 ± 0.2 with 0.1 N HCl. CDAP solution (freshly prepared 100 mg / mL in acetonitrile / WFI, 50/50) was added to reach the appropriate ratio of CDAP / PS (1.5 / 1 w / w). The pH was raised up to the activation pH of 9. 00 ± 0.05 by the addition of 0.5M NaOH and stabilized at this pH until the addition of ADH. After 3 minutes, the ADH was added until reaching the appropriate ADH / PS ratio (8.9 / 1 w / w); the pH was adjusted to the coupling pH of 9.0. The solution was left for 1 hour under pH regulation. The PSAH derivative was concentrated and diafiltered. Coupling PhtD was added at 10 mg / mL in 0.2M NaCl to the PS22FAH derivative in order to achieve a PhtD / PS22FAH ratio of 4/1 (w / w). The pH was adjusted to 5.0 ± 0.05 with HCl. The EDAC solution (20 mg / mL in 0.1 M Tris-HCl, pH 7.5) was added manually in 10 min (250 μL / min) until reaching 1 mg of EDAC / mg of PS22FAH. The resulting solution was incubated for 150 min (although it was also used at 60 min) at 25 ° C under agitation and pH regulation. The solution it was neutralized by the addition of 1 M Tris-HCl with pH 7.5 (1/10 of the final volume) and left 30 min at 25 ° C. Before elution in Sephacryl S400HR, the conjugate was clarified using a 5μm Minisart filter. The resulting conjugate has a final PhtD / PS ratio of 4.1 (w / w), a free PS content of less than 1% and an antigenicity (a-PS / a-PS) of 36.3% and anti-PhtD antigenicity of 7.4 %. 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. Using the different molecular sizes of the reaction components, PS-PD, PS-TT, PS-PhtD, PS-pneumolysin or PS-DT conjugates are eluted first, followed by free PS, then by free PD or by free DT, and finally DMAP and other salts (NaCl, glycine). Fractions containing conjugates are detected by UV280nm- The fractions are grouped according to their Kd, sterilized by filtration (0.22 μm) and stored at + 2-8 ° C. The proportions of PS / Protein in the conjugate preparations were determined. Specific activation / coupling / extinction conditions of PS conjugates of S. pneu / non / ae-Protein D / TT / DT / PhtD / Ply When "μfluid" appein a column header, this indicates that the saccharide was sized by microfluidization before conjugation. The sizes of saccharides after microfluidization are given in Table 2. Table 1. Specific conditions of activation / coupling / extinction of conjugates PS of S. pneumon / ae-Protein D / TT / DT / PhtD / Ply Note: pH a.c.q corresponds to pH for activation, coupling and extinction, respectively. Characterization: Each conjugate was • characterized and met the specifications described in Table 2. The polysaccharide content (μg / mL) was measured by the Resorcinol test and the protein content (μg / mL) by the Lowry test. The final PS / PD ratio (w / w) is determined by the proportion of the concentrations. Free polysaccharide content (%): The free polysaccharide content of conjugates maintained at 4 ° C or stored for 7 days at 37 ° C was determined in the supernatant obtained after incubation with saturated ammonium sulfate carrier protein antibodies, followed by centrifugation. An a-PS / a-PS ELISA was used for the quantification of free polysaccharide in the supernatant. The absence of conjugate it was also controlled by a carrier protein ELISA a / PS a. Antigenicity: The antigenicity in the same conjugates was analyzed in a sandwich ELISA where the capture and detection of antibodies were PS a and Protein a respectively.

Free protein content (%): The unconjugated carrier protein can be separated from the conjugate during the purification step. The residual free protein content was determined using size exclusion chromatography (TSK 5000-PWXL) followed by UV detection (214 nm). The elution conditions allowed the separation of the free carrier protein and the conjugate. The content of free protein in the mother conjugate was then determined against a calibration curve (from 0 to 50 μg / mL of carrier protein). The free carrier protein in% was obtained as follows:% free carrier = (free carrier (μg / mL) / (Total concentration of corresponding carrier protein measured by Lowry (μg / mL) * 100%) Stability: Distribution was measured molecular weight (Kav) and stability on a HPLC-SEC with gel filtration (TSK 5000-PWXL) for conjugates maintained at 4 ° C and stored 7 days at 37 ° C. Characterization 10/11/13/14 -valent is given in Table 2 (see commentary at the end of the table.) Protein conjugates can be adsorbed onto aluminum phosphate and pooled to form the final vaccine.

Concluding: Immunogenic conjugates have been produced, which have proven to be the components of a promising vaccine. Table 2 - Characteristics of the conjugates * Size of PS after microfluidization of natural PS. A 10-valent vaccine was prepared by mixing conjugates of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (for example in a dose of 1, 3, 1, 1, 1, 1, 1, 3, 3, 1 μg of saccharide, respectively per dose for human). An 11-valent vaccine was made by also adding the serotype 3 conjugate of Table 5 (for example with 1 μg of saccharide per dose for human). A 13-valent vaccine was made by also adding the above 19A and 22F conjugates (with 22F either directly linked to PhtD, or alternatively by an ADH linker) (for example, in a dose of 3 μg of each saccharide per dose for human ). A 14-valent vaccine can be made by adding in addition the serotype 6A conjugate above (for example in a dose of 1 μg of saccharide per dose for human). Example 3: Evidence that the inclusion of Haemphilus influenzae protein D in an immunogenic composition of the invention may provide improved protection against acute otitis media (AOM).

Study design The study used a 1 1 Pn-PD vaccine - containing serotypes 1, 3, 4, 5, 6B, 7F, 9V, 1 4, 1 8C, 1 9F and 23F, each conjugated to Protein D of H. influenzae (refer to Table 5 in Example 4). Subjects were randomized into two groups to receive four doses of either the 1 1 Pn-PD vaccine or Havrix vaccine at approximately 3, 4, 5 and 1 2-1 5 months of age. All subjects received I nfanrix-hexa vaccine (DTPa-H BV-I PV / Hib) from GSK Biologicals concomitantly at 3, 4 and 5 months of age. The infanrix-hexa is a combination of Ped iarix and Hib mixed before administration. Follow-up of the efficacy for the analysis "according to the protocol" began 2 weeks after the administration of the third dose of vaccine, and continued until 24-27 months of age. The nasopharyngeal carriage of S. pneumoniae and H. influenzae was evaluated in a selected subset of subjects. The parents were advised to consult the investigator if their child was sick, had an earache, spontaneous perforation of the tympanic membrane, or spontaneous discharge through the ear. If the investigator suspected an episode of AOM, the child was immediately referred to an ear, nose, and throat specialist (ENT) for confirmation of the diagnosis. The clinical diagnosis of AOM was based either on the visual appearance of the tympanic membrane (ie, redness, lumps, loss of light reflex) or the presence of fluid effusion in the middle ear (demonstrated by simple pneumatic otoscopy or by microscope). In addition, at least two of the following signs and symptoms must be present: ear pain, ear discharge, hearing loss, fever, lethargy, irritability, anorexia, vomiting or diarrhea. If the ENT specialist confirmed the clinical diagnosis, a fluid sample from the middle ear was collected by tympanocentesis for bacteriological analysis. For subjects with repeated visits to the disease, it was considered that a new AOM episode had started if more than 30 days had elapsed since the beginning of the previous episode. In addition, an episode of AOM was considered a new bacterial episode if the isolated bacterium / serotype differed from the previous isolate, whatever the interval between the two consecutive episodes. RESULTS OF THE TEST A total of 4968 children were recruited, 2489 in the group of 1 1 Pn-PD and 2479 in the control group. There were no large differences in demographic characteristics or risk factors between the two groups. Clinical episodes and defi nition of the AOM case During the follow-up period of the procedure, a total of 333 episodes of clinical AOM were recorded in the group of 1 1 PN-PD and 499 in the control group. Table 3 presents the protective efficacy of the vaccine 1 1 Pn-PD vaccine and of both 7-valent vaccines tested 409 and Kil pi et al. Clin I nfect Dis 2003 37: 1 1 55-64) against any episode of AOM and AOM produced by different pneumococcal serotypes, H. influenzae, NTH i and M. catarrhalis. A statistically significant and clinically relevant reduction in 33.6% of the AOM disease burden was achieved with 1 1 Pn-PD, if n not consider the etiology (Table 3). The overall efficacy against AOM episodes due to any of the 1 1 pneumococcal serotypes contained in the 1 1 Pn-PD vaccine was 57.6% (Table 3). Another important finding in the current study is the 35.6% protection provided by the 1 1 Pn-PD vaccine against AOM produced by H. influenzae (and specifically the 35.3% protection provided by NTHi). This finding is of relevant clinical importance, given the increasing importance of H. influenzae as a major cause of AOM in the era of pneumococcal conjugate vaccine. In line with the protection provided against AOM, the 1 1 Pn-PD vaccine also reduced the nasopharyngeal carriage of H. influenzae after the booster dose in the second year of life. These findings are in contrast to previous observations in Finland, where for both 7-valent pneumococcal conjugate vaccines, an increase in AOM episodes was observed due to H. influenzae, (Eskola et al and Kil pi et al) as evidence of etiological replacement. It was not possible to establish a clear correlation between the protection against AOM episodes due to Hi and antibody levels against carrier protein D, since the concentrations of anti-PD post-primary IgG antibody in those vaccinated with 1 1 Pn-PD who remained without episodes of AOM H 1, were essentially the same as the post-primary anti-PD IgG antibody levels measured in those vaccinated with 1 1 Pn-P D who developed at least one episode of AOM Hi during the efficacy monitoring period. However, although no correlation could be established between the biological impact of the vaccine and the immunogenicity of anti-PD IgG post-pri maria, it is reasonable to assume that the PD carrier protein, which is highly conserved among H strains, Influenzae, has contributed to a high degree to the induction of protection against Hi. The effect on the AOM disease was accompanied by an effect on nasopharyngeal carriage that was of similar magnitude for the vaccine with serotype of pneumococci and H. influenzae (figure 1). This network of naso-pharyngeal carriage of H. influenzae in PD conjugate vaccines 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 with the anti-PD IgG immune responses measured by EL I SA. In a subsequent experiment an otitis media model was used in chinchilla with serum pools of infants immunized with the formulation 1 1 -valent of this example or with the 1-valent vaccine of Example 2 (see also Table 1 and 2 and the comments below them). Both groups induce a significant reduction in the percentage of animals with otitis media against the pre-immunized serum group. There is no significant difference between the groups immunized with 1 0 and 1 1 -valent. This shows that both vaccines have a similar potential to induce protection against otitis media produced by H. influenzae not classifiable in this model. Table 3 NP = Not published, N = number of subjects in the ATP efficacy cohort; n = number of episodes Pneumococcal vaccine serotypes for 11 Pn-PD = 11 serotypes, for Prevnar and 7v-0MP = 7 serotypes MEF = Fluid in the middle ear Example 4: Selection of carrier protein for serotype 19F ELISA analysis used The ELISA method for the inhibition of 22F was based essentially on an analysis proposed in 2001 by Concepción and Frasch and was reported by Henckaerts et al. 2006, Clinical and Vaccine Immunology 13: 356-360. Briefly, pneumococcal polysaccharides were mixed with methylated human serum albumin and adsorbed onto Nunc Maxisorp ™ microtiter plates (Roskilde, DK) of high degree of fixation, overnight at 4 ° C. Plates were blocked with 10% fetal bovine serum (FBS) in PBS for 1 hour at room temperature with shaking. Serum samples were diluted in PBS containing 10% FBS, 10 μg / mL cell wall polysaccharide (SSI) and 2 μg / mL pneumococcal polysaccharide serotype 22F (ATCC), and further diluted in the microtiter plates. with the same regulator. An internal reference calibrated against the 89-SF standard serum using the IgG concentrations specific for the 89-SF serotype was treated in the same way and included in each plate. After washing, the bound antibodies were detected using peroxidase-conjugated human anti-lgG monoclonal antibody (Stratech Scientific Ltd., Soham, UK) diluted in 10% FBS (in PBS), and incubated for 1 hour at room temperature with shaking. The color was revealed using the equipment with substrate for enzyme immunoassay with ready-to-use tetramethylbenzidine one-component (BioRad, Hercules, CA, US) in the dark at room temperature. The reaction was stopped with 0.18 M H2SO4, and the optical density was read at 450 nm. The concentrations of serotype-specific IgG (in μg / mL) in the samples were calculated by reference to points of optical density within defined limits for the internal reference serum curve, which was modeled by a logistic equation of 4 parameters calculated with SoftMax Pro ™ software (Molecular Devices, Sunnyvale, CA). The limit for the ELISA was 0.05 μg / mL of IgG for all serotypes, taking into account the limit of detection and the limit of quantification. Opsonophagocytosis Analysis At the WHO consultation meeting in June 2003, it was recommended to use an OPA analysis as established in Romero-Steiner et. to the. Clin Diagn Lab Immunol 2003 10 (6): pp1019-1024. This procedure was used to analyze the OPA activity of the serotypes in the following tests. Preparation of coniugados In the studies 11 Pn-PDyDi-001 and 11 Pn-PDyDi-007, three formulations were included for 11-valent vaccine (Table 4) in the is μg e po sac r o is a a conga or with diphtheric oxo e (19F-DT) instead of 1 μg of polysaccharide conjugated with protein D (19F-PD). The conjugation parameters for the studies 11 Pn-PD, 11 Pn-PD and Di-001 and 11 Pn-PD and Di-007 are described in Tables 5, 6 and 7 respectively. Anti-pneumococcal antibody responses or OPA activity against 19F serotype one month after primary vaccination with these 19F-DT formulations are shown in Tables 8 and 9 respectively. Table 10 shows the concentrations of 22F-ELISA antibody and the percentages of subjects who reached the threshold of 0.2 μg / mL before and after booster vaccination with 23-valent simple polysaccharide. It was shown that opsonophagocytic activity clearly improves for the antibodies induced with these formulations of 19F-DT, as shown by the higher rates of seropositivity (opsonophagocytic titers> 1: 8) and GMT OPA one month after primary vaccination (Table 9). One month after booster vaccination with simple 23-valent polysaccharide, the opsonophagocytic activity of 19F antibodies remained significantly better for children sensitized with 19F-DT formulations (Table 11). Table 12 presents immunogenicity data after a booster dose with 11 Pn-PD in young children previously sensitized with 19F-DT or 19F-PD conjugates compared to a 4th. consecutive dose of Prevnat®. Given the reported cases of advancement after the introduction of Prevnar® in the United States, enhanced opsontophagocytic activity against the 19F serotype 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 a low but significant activity of OPA against 19A. Table 4. Pneumococcal conjugate vaccine formulations used in clinical studies.

Table 5. Specific conditions of activation / coupling / extinction of conjugates PS of S. pneumon / 'ae-Protein D / TT / DT Table 6 Specific conditions of activation / coupling / extinction of conjugates PS of S. / opet / mon / ae-Protein D / DT for study 1 1 Pn-PD and Di-001 Table 7 Specific activation / coupling / extinction conditions of PS conjugates of S. pnet / mon / ae-Protein D / DT for study 1 1 Pn-PD and Di-007 Table 8 Percentage of subjects with antibody concent ration 19F > 0.20 ua / mL V average geometric concentrations of antibody 19F ant body (GMC with 95% Cl uo / mU one month after primary vaccination with lug 19F- PD.3ug 19F-DT or Prevnar (2μg 19F-CRM) (Total Cohort) The composition of the different formulations is given in Table 4 Table 9 Percentage of stocks with title OPA 19F > 1: 8 and GMT OPA 19F one month after primary vaccination with 1 ug of 19F-PD.3ug 19F-DT or Prevnar (2ug 19F-CRM) (Total Cohort) The composition of the different formulations is given in Table 4. Table 10 Percentage of subjects with antibody concentration 19F > 0.20 μg / mL and 19F GMC antibody (μg / mL) before and one month after reinforcement with 23-valent simple polysaccharide in children sensitized with 1μg 19F-PD, 3μg 19F-DT or Prevnar (2μg 19F-CRM) (Total cohort ) The composition of the different formulations is given in Table 4. Table 11 Percentage of stocks with title OPA 19F > 1: 8 and GMT OPA 19F before and one month after the reinforcement with 23-valent poly-saccharide simp in sensitized children with 1μq of 19F-PD, 3μg of 19F-DT or P revnar (2μg of 19F-CRM) (Coh orte tota ") The composition of the different formulations is given in Table 4. Table 12 Percentage of subjects with antibody concentrations > 0.2 μg / mL, OPA > 1: 8 v GMC / GMT against 19F pneumococci one month after booster with 11Pn-PD or Prevnar in children sensitized with 1uo de 19F-PD. 3μg of 19F-DT or Prevnar (2ug 19F-CRM) (Total Cohort) The composition of the different formulations is given in Table 4. Table 13 Percentage of subjects with antibody concentrations > 0.2 μg / mL. OPA > 1: 8 and GMC / GMT against 19A pneumococci one month after primary vaccination with 1μg 19F-PD.3μg 19F-DT or Prevnar (2μg 19F-CRM) (Total Cohort) The composition of the different formulations is given in Table 4 Example 5: Experiments with adjuvant in pre-clinical models: impact on the immunogenicity of conjugated 11-valent pneumococcal polysaccharides in elderly Rhesus monkeys To optimize the response elicited to pneumococcal conjugate vaccines in the elderly population, GSK formulated a 11-valent polysaccharide (PS) conjugate vaccine with a novel adjuvant: Adjuvant C - see below. Groups of 5 elderly Rhesus monkeys (14 to 28 years of age) were immunized intramuscularly (IM) on days 0 and 28 with 500 μL of any of the 11-valent PS conjugates adsorbed on 315 μg of AIPO4 or conjugates PS 11-valent mixed with Adjuvant C. In both vaccine formulations, the conjugated PS 11-valent conjugates were each composed of the 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 of the dose corresponding to the human dose of the conjugate vaccine (5 μg of each saccharide per dose for human except for 6B [10 μg]) according to the conditions of Table 6 (Example 4) ), except that 19F was prepared according to the following CDAP process conditions: saccharide sized at 9 mg / mL, PD at 5 mg / mL, an initial PD / PS ratio of 1.2 / 1, 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. The levels of IgG for ELISA Anti-PS and the titles of opsono-phagocytosis were administered in sera collected on day 42. The frequencies of B cells of memory Anti-PS3 were measured by Elispot of peripheral blood cells collected on day 42. According to the results shown hereinafter, Adjuvant C significantly improved the immunogenicity of PS 1 1 -valent conjugates against conjugates with AI PO 4 in elderly monkeys. The novel adjuvant improved the IgG responses to PS (Figure 1) and the antibody titers of opsono-phagocytosis (Table 1 4). There is also supporting evidence that the frequency of memory-specific B cells for PS3 is increased by the use of Adjuvant C (Figure 2). Table 14. Immunogenicity of conjugate in elderly rhesus monkeys (post-l opsonophagocytosis titers) The isot of B cells The principle of the analysis is based on the fact that B memory cells mature in plasma cells in vitro after of CpG culture for 5 days. Antigen-specific plasma cells generated in vitro can be easily detected and therefore can be enumerated using elispot B cell analysis. The amount of specific plasma cells is a mirror of the frequency of B-cell recall at the start of the culture. Briefly, in vitro-generated plasma cells are incubated in culture plates coated with antigen. The antigen-specific plasma cells form antibody / antigen spots, which are detected by conventional immunoenzymatic procedure and listed as B-cells of recall. In the present study, polysaccharides have been used to coat culture plates in order to list the respective B cells of remembrance. The results are expressed as the frequency of B memory cells within one million B-memory cells. The study shows that Adjuvant C may be able to overcome the problem known as PS3 booster capacity (see 5th International Symposium on Pneumococcal and Pneumococcal Diseases, April 2-6 2006, Al ice Springs, Central Australia. responses against a serotype 3 pneumococcal conjugate Schuerman L, Prymula R, Poolman J. Abstract book p 245, PO10.06). Eiem plo 6. Effectiveness of detoxified pneumolysis (d Ply) as a carrier protein to improve the immunity of PS 19F in young Balb / c mice Groups of 40 female Balb / c mice (4 weeks old) were immunized IM on days 0, 14 and 28 with 50 μL of either PS 4-valent single or dPIy-conjugated PS 4- valent, both mixed with Adjuvant C. Both vaccine formulations were composed of 0.1 μg (amount of saccharide) from each of the following PS: PS8, PS12F, PS19F and PS22F. Anti-PS IgG levels for ELISA were dosed in sera collected on day 42. The anti-PS19F response, shown as an example in Figure 3, was greatly improved in mice to which the conjugated dPly were administered. -valent compared to mice immunized with simple PS. The same improvement was observed for anti-PS8, 12F and 22F IgG responses (data not shown). Example 7. Effectiveness of Protein D Pneumococcal Histidine Histidine in triad (PhtD) as a carrier protein 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 in on days 0, 14 and 28 with 50 μL of either 4-valent simple PS or 4-valent conjugated PhtD-PS, both mixed with Adjuvant C. Both vaccine formulations were composed of 0.1 μg (amount of saccharide) from each of the following PS: PS8, PS12F, PS19F and PS22F. Levels of Anti-PS IgG for ELISA were dosed in sera collected on the day 42. The anti-PS22F response, shown as an example in Figure 4, was markedly improved in mice given the 4-valent PhtD conjugates compared to mice immunized with the simple PS. The same improvement was observed for the anti-PSG, 12F and 19F IgG responses (data not shown). Example 8. Immunogenicity in elderly C57BI mice of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD Groups of 30 elderly C57BI mice (>69 weeks of age) were immunized IM on days 0, 14 and 28 with 50 μL of either 11-valent PS conjugates or 13-valent PS conjugates, both mixed with Adjuvant C (see below). The 11-valent vaccine formulation was composed of 0.1 μg of saccharide from 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 the 11-valent vaccine described under Table 2). The 13-valent vaccine formulation also contained 0.1 μg of PS19A-dPly and PS22F-PhtD conjugates (see Table 1 and comment on the 13-valent vaccine described under Table 2 [using directly conjugated 22F]). In group 2 and 4 the carrier pneumolysin was detoxified with treatment with GMBS, in group 3 and 5 this was done with formaldehyde. In groups 2 and 3 PhtD was used for the conjugate PS 22F, in groups 4 and 5 ase used a fusion of PhtD_E (construction VP147 of WO 03/054007). In group 6, 19A was conjugated with diphtheria toxoid and 22F with protein D Anti-PS19A and the 22F IgG levels were dosed for ELISA in individual sera collected on day 42. The ELISA of the IgG response generated for the other PSs was measured in grouped sera. 19A-dPly and 22F-PhtD administered within the formulation for 13-valent conjugate vaccine proved to be immunogenic in elderly C57BI mice (Table 15). The immune response induced against the other SPs did not negatively impact the mice given the 13-valent formulation compared to those immunized with the 11-valent formulation. Table 15. Immunogenicity of PS in elderly C57BI mice (post-lll IgG levels) Example 9. Immunogenicity in young Balb / c mice of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD Groups of 30 young Balb / c mice (4 weeks of age) were immunized IM on days 0, 14 and 28 with 50 μL of 11-valent PS conjugates or 13-valent PS conjugates, both mixed with Adjuvant C (see below). The 11-valent vaccine formulation was composed of 0.1 μg of saccharide from 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 commentary on the 11-valent vaccine described under Table 2). The 13-valent vaccine formulation also contained 0.1 μg of PS19A-dPly and PS22F-PhtD conjugates (see Table 1 and comment on the 13-valent vaccine described under Table 2 [using directly conjugated 22F]). In group 2 and 4 the carrier pneumolysin was detoxified with treatment with GMBS, in group 3 and 5 this was done with formaldehyde. In groups 2 and 3 PhtD was used for the PS 22F conjugate, in groups 4 and 5 a fusion of PhtD E (the VP 147 construction of WO 03/054007) was used. In group 6, 19A was conjugated with diphtheria toxoid and 22F with protein D. Anti-PS19A and 22F IgG levels for ELISA were dosed in individual sera collected on day 42. The IgG response to ELISA generated for the other SPs was measured in pooled sera. The 1 9A-d Ply and the 22F-PhtD administered within the formulation for 1 3-valent conjugate vaccine proved to be immunogenic in young Balb / c mice (Table 1 6). The immune response induced against the other SPs was not negatively impacted in mice given the 3-valent formulation as compared to those immunized with the 1-valent formulation. Table 16. Immunogenicity of PS in young Bal b / c mice (levels of post-ll l IgG) Example 10. Immunogenicity in Guinea pigs of PS 13-valent conjugates containing 19A-dPly and 22F-PhtD Groups of 20 young guinea pigs (Hartley breed, 5 weeks old) were immunized IM on days 0, 14 and 28 with 125 μL of any PS 11-valent conjugates or PS 13-valent conjugates, both mixed with Adjuvant C (see below). The 11-valent vaccine formulation was composed of 025 μg of saccharide from 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 commentary on the 11-valent vaccine described under Table 2). The 13-valent vaccine formulation also contained 0.1 μg of conjugated PS19A-dPly and PS22F-PhtD (see Table 1 and comment on the 13-valent vaccine described under Table 2 [using directly conjugated 22F]). In group 2 and 4 the carrier pneumolysin was detoxified with treatment with GMBS, in group 3 and 5 this was done with formaldehyde. In groups 2 and 3 PhtD was used for the PS 22F conjugate, in groups 4 and 5 a PhtD_E fusion (the VP147 construction of WO 03/054007) was used. In group 6, 19A was conjugated with diphtheria toxoid and 22F with protein D. The levels of IgG Anti-PS19A and 22F were dosed for ELISA in individual sera collected on day 42. The IgG response generated for the other PS was measured in grouped sera. Table 17. Immunogenicity of PS in young Balb / c mice (post-lll IgG levels) Example 11: Elaborated and tested formulations a) The following formulations were made (using the 13-valent vaccine from Table 1 and serotype 3 from Table 5 - see the comment on the 14-valent vaccine described under Table 2 [using directly -conjugated 22F or by means of an ADH linker]). The saccharides are formulated with aluminum phosphate and 3D-MPL as shown below. b) The same saccharide formulation is aided with adjuvant with each of the following adjuvants: - The concentration of the components of the emulsion for 500 μL of dose is shown in the table below.

Ingredients Adjuvant A1 Adjuvant A2 Adjuvant A3 250μl emulsion of 125μl emulsion of 50μl emulsion of oil in water oil in water oil in water alpha 11.88mg 5.94mg 2.38mg Tocopherol Squalene 10Jmg 5.35mg 2.14mg Tween 80 4.85mg 2.43mg 0. 97mg Ingredients Adjuvant A4 Adjuvant A5 Adjuvant A6 Adjuvant A7 250μl emulsion 250μl emulsion 125μl emu Isión 50μl emulsion of oil in oil in oil in oil in water water water water alfa 11.88mg 11.88mg 5.94mg 2.38mg Tocoferol Squalene 10Jmg 10Jmg 5.35mg 2.14mg Tween 80 4.85mg 4.85mg 2.43mg 0.97mg 3D-MPL 50μg 25μg 25μg 10μg c) The saccharides are also formulated with two adjuvants based on liposomes: Composition of Quantitative Qualitative Adjuvant B1 (per dose of 0.5 mL) Liposomes: DOPC 1 mg cholesterol 0.25 mg 3D MPL 50 μg QS21 50 μg KH2PO4 T 3.1 24 mg Shock absorber Na2H PO4 T 0.290 mg NaCI shock absorber 2.922 mg (100 mM) WFI sufficient quantity for 0.5 ml of solvent pH 6.1 1. Total concentration of PO = 50 mM Composition of Quantitative Quantitative Adjuvant B2 (per dose of 0.5 mL) Liposomes: DOPC 0.5 mg cholesterol 0.125 mg 3DMPL 25 μg QS 21 25 μg KH2PO4 -, 3.124 mg Shock absorber Na2HPO41 0.290 mg Shock absorber NaCl 2.922 mg (100 mM) WFI sufficient quantity for 0.5 ml of solvent pH 6.1 d) The saccharides are also formulated with Adjuvant C (see above for other compositions where this adjuvant has been used): Qualitative Quantitative (per dose of 0.5 mL) Oil in water emulsion: 50 μL - squalene 2.136 mg - a-tocopherol 2.372 mg - Tween 800.97 mg - cholesterol 0.1 mg 3DMPL 50 μg QS21 50 μg KH2PO t? .470 mg Shock absorber Na2HPO4 t? .219 mg Shock absorber NaCl 4.003 mg (137 mM) KCl 0.101 mg (2.7 mM) WFI sufficient amount for 0.5 ml of solvent pH 6.8 Example 12. Impact of conjugation chemistry in the immunogenicity of the 22F-PhtD conjugate in Balb / c mice Groups of 30 female Balb / c mice were immunized intramuscularly (IM) on 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 μg of saccharide / conjugate for PS 4, 18C, 19A, 19F and 22F and 0.1 μg of saccharide / conjugate for the other PS). PS 18C was conjugated with tetanus toxoid, 19F with diphtheria toxoid, 19A with PIy detoxified with formaldehyde, 22F with PhtD and the other PS with PD. Two formulations, consisting of either 22F-PhtD prepared by direct CDAP chemistry or 22F-AH-PhtD, were compared.

(PS derived from ADH). See Example 2, Table 1 and the comment under Table 2 for the characteristics of the 13-valent vaccine prepared either with 22F directly conjugated or by means of a ADH separator. The vaccine formulations were supplemented with adjuvant C. Anti-PS22F IgG levels for ELISA and opsono-phagocytosis titres were measured in sera collected on day 42. It was shown that 22F-AH-PhtD is much more immunogenic than 22F-PhtD both in terms of the levels of IgG (figure 5) and of the opsono-phagocytic titles (figure 6). Example 13. Impact of new adjuvants on the immunogenicity of conjoined PS of Streptoccoccus pneumoniae Groups of 40 female Balb / c mice were immunized IM on 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 μg / conjugate for PS 4, 18C, 19A, 19F and 22F and 0.1 μg / conjugate for the other PS). PS 18C was conjugated with tetanus toxoid, 19F with Diphtheria Toxoid, 19A with PIy detoxified with formaldehyde, 22F with PhtD and the other PS with PD. See Example 2, Table 1 and the comment under Table 2 for the characteristics of the 13-valent vaccine prepared with 22F directly conjugated. Four formulations were compared, complemented with AIPO4, adjuvant A1, adjuvant A4 or adjuvant A5. The levels of IGG Anti-PS, PIy, PhtD and PD were measured for ELISA in sera collected on day 42 and grouped by group. The following ratio was calculated for each antigen: IgG level induced with the new adjuvant analyzed / level of IgG induced with AIPO. All the new adjuvants tested improved at least 2-fold the immune responses to the 13-valent conjugates compared to the classical AIPO formulation (Figure 7). Example 14. Protective efficacy of a detoxified PhtD / Ply group in a model of pneumococcal pneumonia in mono Groups of 6 Rhesus monkeys (3 to 8 years of age), selected as having the lowest pre-existing anti-19F antibody levels , were immunized intramuscularly on days 0 and 28 with any of the 11-valent PS conjugates (ie 1 μg of PS 1, 3, 5, 6B, 7F, 9V, 14 and 23F, and 3 μg of PS 4 , 18C and 19F [saccharide]) or PhtD (10μg) + PIy detoxified with formaldehyde (10 μg) or the adjuvant alone. PS 18C was conjugated with tetanus toxoid, 19F with diphtheria toxoid and the other PS with PD. See Example 2, Table 1 and the comment under Table 2 to determine the characteristics of the 11-valent vaccine. All formulations were supplemented with adjuvant C. Pneumococci of type 19F (5,108 cfu) were inoculated in the right lung on day 42. Colonies were counted in broncho-alveolar lavages collected on days 1, 3 and 7 after exposure . The results were expressed as the number of animals per group, either dead, with colonized lung or out of danger on day 7 after exposure. As shown in Figure 8, a good close exposure to statistical significance was obtained (weights at the low number of animals used) with the 11-valent conjugates and with the PhtD + dPly group (p <0.12, Fisher's exact test ) compared to the group with the adjuvant alone. Example 15. Impact of conjugation guímica on the response of the anti-PhtD antibody and the protective efficacy against a type 4 exposure induced by coniugados 22F-PhtD. Groups of 20 female OF1 mice were immunized intramuscularly on days 0 and 14 with 3 μg of 22F-PhtD (prepared by direct CDAP chemistry) or 22F-AH-PhtD (PS derived from ADH), 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 complemented with adjuvant C. Anti-PhtD IgG levels for IgG ELISA were measured in sera collected on day 27. Mice were exposed intranasally with 5,106 cfu type 4 pneumococci on day 28 (ie a pneumococcal serotype n). potentially covered by the PS present in the vaccine formulation tested). Induced mortality was monitored until day 8 after exposure. 22F-AH-PhtD induced a significantly higher anti-PhtD IgG response and better protection against exposure to type 4 than 22F-PhtD.

Claims (136)

1. An immunogenic composition for children containing a multivalent Streptococcus pneumoniae vaccine containing 2 or more (for example 7, 8, 9, 10, 11, 12, 13, 14, 15) conjugated capsular saccharides of different serotypes, wherein the composition contains a saccharide conjugate of serotype 22F.

2. The immunogenic composition of claim 1 containing capsular saccharide conjugates of S. pneumoniae of serotypes 19A and / or 19F.

The immunogenic composition of claim 1 or 2, containing capsular saccharide conjugates of S. pneumoniae of serotypes 19A and 19F wherein 19A is conjugated to a carrier protein that is a first bacterial toxoid and 19F is conjugated to a second toxoid bacterial.

4. The immunogenic composition of claim 3 wherein the first bacterial toxoid is a protein different from the second bacterial toxin.

The immunogenic composition of claim 3 or 4 wherein the first bacterial toxoid is selected from the group consisting of tetanus toxoid, diphtheria toxoid, CRM 197, pertussis toxoid, bacterial cytolysin, and pneumolysin.

6. The immunogenic composition of any of claims 3-5 wherein the second bacterial toxoid is selected from the group consisting of tetanus toxoid, toxoid diphtheria, CRM197, pertussis toxoid, bacterial cytolysin and pneumolysin

7. The immunogenic composition of any of claims 3-6 wherein the first bacterial toxoid is pneumolysin

8. The immunogenic composition of any of claims 3-7 wherein the second Bacterial toxoid is diphtheria toxoid.

9. The immunogenic composition of any of claims 1-8 which further contains conjugates of capsular saccharides of S. pneumoniae 4, 6B, 9V, 14, 18C and 23F.

10. The immunogenic composition of claims 1-9 which further contains conjugates of capsular saccharides of S. pneumoniae 1, 5 and 7F.

11. The immunogenic composition of claims 1-10 further containing a conjugate 3 of capsular saccharide of S. pneumoniae.

12. The immunogenic composition of claims 1-1 1 further containing a 6A conjugate of capsular saccharide of S. pneumoniae.

The immunogenic composition of any of claims 1-12 wherein 2 different carrier proteins are separately conjugated to at least 2 different serotypes of capsular saccharide of S. pneumoniae.

14. The immunogenic composition of any of the claims 1-12 wherein 3 different carrier proteins are conjugated separately with at least 3 different serotypes of capsular saccharide of S. pneumoniae.

15. The immunogenic composition of any of claims 1-12 wherein 4 different carrier proteins are separately conjugated with at least 4 different serotypes of capsular saccharide of S. pneumoniae.

16. The immunogenic composition of any of claims 1-12 wherein 5 or more different carrier proteins are separately conjugated with at least 5 different serotypes of capsular saccharide of S. pneumoniae.

The immunogenic composition of claims 13-16 which contains 2 or more of the carrier proteins selected from the following list: tetanus toxoid, diphtheria toxoid, pneumolysin, Protein D and PhtD or fusion proteins thereof.

18. The immunogenic composition of claims 1-17 containing capsular saccharide 1 of S. pneumoniae conjugated with protein D.

19. The immunogenic composition of claims 1-18 containing capsular saccharide 3 of S. pneumoniae conjugated with protein D, pneumolysin or PhtD or fusion protein of them.

20. The immunogenic composition of claims 1-19 containing capsular saccharide 4 of S. pneumoniae conjugated to protein D.

21. The immunogenic composition of claims 1-20 containing capsular saccharide 5 of S. pneumoniae conjugated with protein D.

22. The immunogenic composition of claims 1-20 containing capsular saccharide 6B of S. pneumoniae conjugated with protein D.

23. The immunogenic composition of claims 1-22 containing 7F capsular saccharide of S. pneumoniae conjugated with protein D

24. The immunogenic composition of claims 1 -23 containing 9V capsic saccharide of S. pneumoniae conjugated with protein D.

25. The immunogenic composition of claims 1-24 which also contains 1 4 capsular saccharide of S. pneumoniae. conjugate with protein D

26. The immunogenic composition of claims 1 -25 containing 23F capsular saccharide of S. pneumoniae conjugated with protein D

27. The immunogenic composition of claims 1-26 containing capsular saccharide 18C of S. pneumoniae conjugated to Tetanus toxoid

28. The immunogenic composition of claims 1-27 containing 1 9A capsic saccharide of S. pneumoniae conjugated to pneumolysin

29. The immunogenic composition of claims 1 -28 containing 22F capsular saccharide conjugated with S. pneumoniae PhtD or fusion protein of them.

30. The immunogenic composition of claims 1-29 containing capsular saccharide 6A of S. pneumoniae conjugated with pneumolysin or a protein of H. influenzae, optionally protein D or PhtD or fusion protein thereof.

31. The immunogenic composition according to any preceding claim which contains a 19A capsular saccharide conjugated directly to the carrier protein.

32. The immunogenic composition of any of claims 1-30 wherein 19A capsular saccharide is conjugated to the carrier protein by a linker.

33. The immunogenic composition of claim 32 wherein the binder is ADH.

34. The immunogenic composition of claim 32 or 33 wherein the binder is bound to the carrier protein by carbodiimide chemistry, preferably using EDAC.

35. The immunogenic composition of any of claims 31-34 wherein the saccharide 19A is conjugated to the carrier protein or to the linker using CDAP chemistry.

36. The immunogenic composition of any of claims 1-35 containing a conjugated serotype 19A wherein the ratio of carrier protein to saccharide 19A is between 5: 1 and 1: 5, 4: 1 and 1: 1 or 3.5 : 1 and 2.5: 1 (p / p).

37. The immunogenic composition according to any preceding claim containing a 19F capsular saccharide conjugated directly with the carrier protein.

38. The immunogenic composition of any of claims 1-36 wherein 19F capsular saccharide is conjugated to the carrier protein by a linker.

39. The immunogenic composition of claim 38 wherein the binder is ADH.

40. The immunogenic composition of claim 38 or 39 wherein the binder is bound to the carrier protein by carbodiimide chemistry, preferably using EDAC.

41. The immunogenic composition of any of claims 37-40 wherein the saccharide 19F is conjugated to the carrier protein or to the linker using CDAP chemistry.

42. The immunogenic composition of any of claims 1-41 containing a 19F saccharide conjugate, wherein the ratio of carrier protein to saccharide 19F is between 5: 1 and 1: 5, 4: 1 and 1: 1 or 2: 1 and 1: 1 (p / p), or 1.5: 1 and 1.4: 1.

43. The immunogenic composition according to any preceding claim containing a 22F capsular saccharide conjugate directly with the carrier protein.

44. The immunogenic composition of any of claims 1-42 containing 22F capsular saccharide conjugated to the carrier protein by a linker.

45. The immunogenic composition of claim 44 wherein the binder is ADH.

46. The immunogenic composition of claim 44 or 45 wherein the binder is attached to the carrier protein by carbodiimide chemistry, preferably using EDAC.

47. The immunogenic composition of any of claims 43-46 wherein the saccharide 22F is conjugated to the carrier protein or to the linker using CDAP chemistry.

48. The immunogenic composition of any of claims 1-47 containing a saccharide conjugate 22F, wherein the ratio of carrier protein to saccharide 22F is between 5: 1 and 1: 5, 4: 1 and 1: 1 or 2: 1 and 1: 1 (p / p).

49. The immunogenic composition of any preceding claim containing a saccharide conjugate 19A, wherein the average size (e.g. Mw) of the saccharide 19A is greater than 100 kDa

50. The immunogenic composition of claim 49 wherein the average size (per example Mw) of saccharide 19A is between 50 and 800 kDa, 110 and 700 kDa, 110-300, 120-200, 130-180, or 140-160 kDa.

51. The immunogenic composition of claim 49 or 50 wherein the saccharide 19A is a natural polysaccharide or is sized by a factor of not more than x5.

52. The immunogenic composition of claims 49-51 wherein the saccharide 19A has been sized by microfluidization.

53. The immunogenic composition of any preceding claim containing a saccharide conjugate of serotype 19A, in wherein the dose of the saccharide conjugate 19A is between 1 and 10 μg, 1 and 5 μg, or 1 and 3 μg of saccharide.

54. The immunogenic composition of claim 53 wherein the dose of the saccharide conjugate 19A is 3 μg of saccharide.

55. The immunogenic composition of any preceding claim containing a saccharide conjugate 22F, wherein the average size (for example Mw) of saccharide 22F is greater than 100 kDa

56. The immunogenic composition of claim 55 wherein the average size ( for example Mw) of saccharide 22F is between 50 and 800 kDa, 110 and 700 kDa, 110-300, 120-200, 130-180, or 150-170 kDa.

57. The immunogenic composition of claim 55 or 56 wherein the saccharide 22F is a natural polysaccharide or is dimensioned by a factor of not more than x5.

58. The immunogenic composition of claims 55-57 wherein the saccharide 22F has been sized by microfluidization.

59. The immunogenic composition of any preceding claim containing a saccharide conjugate of serotype 22F, wherein the dose of saccharide conjugate 22F is between 1 and 10 μg, 1 and 5 μg, or 1 and 3 μg of saccharide

60. The immunogenic composition of claim 59 wherein the dose of saccharide conjugate 22F is 3 μg of saccharide.

61. The immunogenic composition of any claim precedent where the average size (for example Mw) of the saccharides is greater than 50 kDa, for example, 50-1600, 80-1400, 100-1000, 150-500, or 200-400 kDa.

62. The immunogenic composition according to claim 61 containing serotype 1 (saccharide conjugate) having an average saccharide size (for example Mw) of between 100-1000, 200-800, 250-600, or 300 and 400 kDa .

63. The immunogenic composition according to claim 61 or 62 containing serotype 4 (saccharide conjugate) having an average saccharide size (for example Mw) of between 50-500, 60-300, 70-200, or 75 and 125 kDa.

64. The immunogenic composition according to claims 61-63 containing serotype 5 (saccharide conjugate) having an average saccharide size (for example Mw) of between 100-1000, 200-700, 300-500, or 350 and 450 kDa.

65. The immunogenic composition according to any of claims 61 to 64 containing serotype 6B (saccharide conjugate) having an average saccharide size (for example Mw) of between 500-1600, 750-1500, or 1000 and 1400 kDa .

66. The immunogenic composition according to any of claims 61 to 65 containing serotype 7F (saccharide conjugate) having an average saccharide size (for example Mw) of between 50-1000, 100-750, 150-500, or 200 and 300 kDa.

67. The immunogenic composition according to any of claims 61 to 66 containing serotype 9V (saccharide conjugate) having an average saccharide size (for example Mw) of between 50-1000, 100-750, 150-500, 200-400, or 250 and 300 kDa.

68. The immunogenic composition according to any of claims 61 to 67 which contains serotype 14 (saccharide conjugate) having an average saccharide size (for example Mw) of between 50-1000, 100-750, 150-500, or 200 and 250 kDa.

69. The immunogenic composition according to any of claims 61 to 68 containing serotype 23F (saccharide conjugate) having an average saccharide size (eg Mw) of between 500-1500, 700-1300, 800-1100, or 900 and 1000 kDa.

70. The immunogenic composition of any preceding claim containing serotypes 5, 6B and 23F (and optionally 6A) as natural saccharides.

71. The immunogenic composition of any preceding claim wherein the dose of the capsular saccharide conjugates is between 1 and 10 μg, 1 and 5 μg, or 1 and 3 μg of saccharide per conjugate.

72. The immunogenic composition of any preceding claim containing conjugates of serotypes 4, 18C, 19F and 22F (and optionally 19A) in doses of 3 μg of saccharide per conjugate.

73. The immunogenic composition of any preceding claim containing conjugates of serotypes 1, 5, 6B, 7F, 9V, 14 and 23F (and optionally 6A and / or 3) in a dose of 1 μg of saccharide per conjugate.

74. The immunogenic composition of any preceding claim which also contains non-conjugated S. pneumoniae saccharides of serotypes other than the conjugates, such that the amount of unconjugated and conjugated saccharide serotypes is less than 23.

75. The immunogenic composition of any preceding claim which also contains one or more conjugated or unconjugated S. pneumoniae proteins.

76. The immunogenic composition of claim 75 which contains one or more conjugated S. pneumoniae proteins.

77. The immunogenic composition of claim 75 or 76 wherein said one or more S. pneumoniae proteins are selected from the poly triad family in triad (PhtX), family of choline binding protein (CbpX), truncated CbpX. , LytX family, truncated LytX, truncated Truncated-LytX CbpX proteins, detoxified pneumolysin (PIy), PspA, PsaA, Sp 1 28, Sp1 01, Sp 1 30, Sp 1 25 and Sp1 33.

78. The immunogenic composition of claims 75-77 containing pneumolysin.

79. The immunogenic composition of any of claims 75-78 which contains a PhtX protein.

80. The immunogenic composition according to any preceding claim containing pneumolysin as a protein free or carrier.

81. The immunogenic composition according to any preceding claim which contains a PhtX protein as a free or carrier protein.

82. The immunogenic composition of claim 81 wherein said PhtX protein is PhtD or a PhtBD or PhtDE fusion protein.

83. The immunogenic composition according to any preceding claim further comprising an adjuvant.

84. The immunogenic composition of claim 83, wherein the adjuvant contains a liposome carrier.

85. The immunogenic composition of claim 84, wherein the adjuvant contains (per dose of 0.5 mL) 0.1-10 mg, 0.2-7, 0.3-5, 0.4-2, or 0.5-1 mg (for example 0.4-0.6). , 0.9-1.1, 0.5 or 1 mg) of phospholipid (for example DOPC).

86. The immunogenic composition of claim 84 or 85, wherein the adjuvant contains (per dose of 0.5 mL) 0.025-2.5, 0.05-1.5, 0.075-0.75, 0.1-0.3, or 0.125-0.25 mg (for example 0.2- 0.3, 0.1-0.15, 0.25 or 0.125 mg) of sterol (for example cholesterol).

87. The immunogenic composition of claims 84-86, wherein the adjuvant contains (per dose of 0.5 mL) 5-60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of lipid A derivative (for example 3D-MPL).

88. The immunogenic composition of claims 84-87, wherein the adjuvant contains (per dose of 0.5 mL) 5-60, 10- 50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of saponin (for example QS21).

89. The immunogenic composition of claim 83, wherein the adjuvant contains an oil in water emulsion.

90. The immunogenic composition of claim 89, wherein the adjuvant contains (per 0.5 mL dose) 0.5-15, 1-13, 2-11, 4-8, or 5-6mg (eg 2-3, 5). -6, or 10-1 1 mg) metabolizable oil (such as squalene).

91. The immunogenic composition of claim 89 or 90, wherein the adjuvant contains (per dose of 0.5 mL) 0.1-10, 0.3-8,

0. 6-6, 0.9-5, 1-4, or 2-3 mg (for example 0.9-1.1, 2-3 or 4-5 mg) of emulsifier (such as Tween 80).

92. The immunogenic composition of claims 89-91, wherein the adjuvant contains (per 0.5 mL dose) 0.5-20, 1 -15, 2-12, 4-10, 5-7 mg (for example 1 1- 13, 5-6, or 2-3 mg) of tocol (such as alpha tocopherol).

93. The immunogenic composition of claims 89-92, wherein the adjuvant contains (per dose of 0.5 mL) 5-60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of lipid A derivative (for example 3D-MPL).

94. The immunogenic composition of claims 89-93, wherein the adjuvant contains (per dose of 0.5 mL) 0.025-2.5, 0.05-1.5, 0.075-0.75, 0.1 -0.3, or 0.125-0.25 mg (for example 0.2- 0.3, 0.1-0.15, 0.25 or 0.125 mg) of sterol (for example cholesterol).

95. The immunogenic composition of claims 89- 94, wherein the adjuvant contains (per dose of 0.5 mL) 5-60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of saponin (for example QS21).

96. The immunogenic composition of claim 83, wherein the adjuvant contains a metal salt and a lipid derivative A.

The immunogenic composition of claim 96, wherein the adjuvant contains (per dose of 0.5 mL) 100-750, 200-500, or 300-400 μg of Al as aluminum phosphate.

98. The immunogenic composition of claim 96 or 97, wherein the adjuvant contains (per dose of 0.5 mL) 5-60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) of lipid A derivative (for example 3D-MPL).

99. A vaccine kit containing an immunogenic composition according to any of claims 1 to 82 and further comprising for concomitant or sequential administration, an adjuvant as defined in any of claims 83 to 98.

100. A vaccine that contains the immunogenic composition of any one of claims 1 to 98 and a pharmaceutically acceptable excipient.

101. A process for making the vaccine according to claim 100 comprising the step of mixing the immunogenic composition of any of claims 1 to 98 with a pharmaceutically acceptable excipient.

102. A method for immunizing a human host against disease caused by infection with Streptococcus pneumoniae comprising administering to the host an immunoprotective dose of the immunogenic composition of any of claims 1 to 98 or the vaccine of claim 100.

103. The method of the claim 102, wherein the human host is a person of advanced age, and the disease is either or both of pneumonia or invasive pneumococcal disease (IPD).

104. The method of claim 102 or 103, wherein the human host is an elderly person, and the disease is exacerbations of chronic obstructive pulmonary disease (COPD).

105. The method of claim 102, wherein the human host is a child, and the disease is otitis media.

106. The method of claim 102 or 105, wherein the human host is a child, and the disease is meningitis and / or bacteremia.

107. The method of claims 102, 105 or 106, wherein the human host is a child, and the disease is pneumonia and / or conjunctivitis.

108. The immunogenic composition of claims 1-98 or the vaccine of claim 100 for use in the treatment or prevention of disease caused by infection with Streptococcus pneumoniae.

109. A use of the immunogenic composition or vaccine of claims 1 to 98 or vaccine of claim 100 in the manufacture of a medicament for the treatment or prevention of diseases caused by infection with Streptococcus pneumoniae.

110. The use of claim 109, wherein the disease is either or both of pneumonia or invasive pneumococcal disease (IPD) of elderly humans.

111. The use of claim 109 or 110, wherein the disease is exacerbations of chronic obstructive pulmonary disease (COPD) of elderly humans.

112. The use of claim 109, wherein the disease is otitis media of human infants.

113. The use of claim 109 or 1 12, wherein the disease is meningitis and / or bacteremia of human infants.

114. The use of claims 109, 12 or 13, wherein the disease is pneumonia and / or conjunctivitis of human infants.

115. A method for eliciting a protective immune response in children against otitis media comprising administration as separate or combined components, sequentially or concomitantly (i) a immunogenic composition or vaccine according to any of claims 1 to 98 and (ii) Protein D of Haemophilus influenzae whose protein D can be free and / or conjugated.

116. A method to trigger an immune response Protection in children against S. pneumoniae by administering the immunogenic composition or vaccine of any preceding claim.

117. A method for eliciting a protective immune response for the elderly against S. pneumoniae, by administering, in combination, sequentially or concomitantly (i) the immunogenic composition or vaccine of any preceding claim (ii) one or more surface proteins of S. pneumoniae selected from the group consisting of the PhtX family and pneumolysin.

118. The immunogenic composition of claims 1-98 or vaccine of claim 100, which contains saccharide conjugates from at least all of the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F wherein the GMC antibody titer induced against one or more of the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates.

119. The immunogenic composition of claim 118, wherein the GMC antibody titer induced against serotype 4 is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates.

120. The immunogenic composition of claim 1 or 18, wherein the GMC antibody titer induced against serotype 6B is not significantly inferior to that induced by the Prevnar® vaccine in human vaccinates.

121. The immunogenic composition of claims 118-120, wherein the GMC antibody titer induced against serotype 9V is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates.

122. The immunogenic composition of claims 1 18- 121, wherein the GMC antibody titer induced against serotype 14 is not significantly inferior to that induced by the Prevnar® vaccine in human vaccinates.

123. The immunogenic composition of claims 1 18-122, wherein the GMC antibody titer induced against serotype 18C is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates

124. The immunogenic composition of claims 1 - 123, wherein the GMC antibody titer induced against 19F serotype is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates

125. The immunogenic composition of claims 118-124, wherein the GMC antibody titer induced against the 23F serotype is not significantly lower than that induced by the Prevnar® vaccine in human vaccinates

126. The immunogenic composition of claims 118-125 that contains a saccharide conjugate of serotype 3.

127. The immunogenic composition of claims 118-126 that contains a saccharide conjugate of serotype 6A.

128. The immunogenic composition of claims 118- 127 which contains a saccharide conjugate of serotype 19A.

129. The immunogenic composition of claims 118-128 which contains a saccharide conjugate of serotype 22F.

130. An immunogenic composition containing at least four capsular saccharide conjugates of S. pneumoniae containing saccharides of different serotypes of S. pneumoniae wherein at least one saccharide is conjugated to PhtD or fusion protein thereof and the immunogenic composition is capable of trigger an effective immune response against PhtD

131. A method to prevent an elderly human host from having a pneumococcal disease caused by infection with Streptococcus pneumoniae serotype 22F (or reducing its severity) which comprises administering it to a childhood host (or to a child population) ) an immunoprotective dose of the immunogenic composition of any of claims 1 to 98 or the vaccine of claim 100.

132. The method of claim 131, wherein the disease is either or both of pneumonia or invasive pneumococcal disease (IPD).

133. The method of claim 131 or 132, wherein the The disease is exacerbations of chronic obstructive pulmonary disease (COPD).

134. A use of the immunogenic composition or vaccine of claims 1 to 98 or vaccine of claim 100 in the manufacture of a medicament for the prevention or reduction of severity of a disease caused by infection by Streptococcus pneumoniae serotype 22F in elderly patients, wherein a immunoprotective dose of said composition or vaccine is administered to a child (or child population).

135. The use of claim 134, wherein the disease is either or both of pneumonia or invasive pneumococcal disease (IPD) of elderly humans.

136. The use of claim 134 or 135, wherein the disease is exacerbations of chronic obstructive pulmonary disease (COPD) of elderly humans. SUMMARY The present invention belongs to the field of pneumococcal capsular saccharide conjugate vaccines. Specifically, an immunogenic composition for children is provided which includes a multivalent Streptococcus pneumoniae vaccine containing two or more conjugated capsular saccharides of different serotypes, wherein the composition contains a saccharide conjugate of serotype 22F. This vaccine can be used in infant populations to reduce the incidence of pneumococcal disease in later life, such as in the case of CO PD exacerbations and / or PD I exacerbations.