MX2008008139A - Pneumococcal polysaccharide conjugate vaccine - Google Patents

Abstract

The present invention is in the field of pneumococcal capsular saccharide conjugate vaccines. Specifically, a multivalent Streptococcuspneumoniaeimmunogenic composition is provided with various conjugated capsular saccharides from differentS. pneumoniaeserotypes conjugated to 2 or more different carrier proteins, where the composition comprises serotype 19F capsular saccharide conjugated to diphtheria toxoid (DT) or CRM197, optionally wherein 19F is the only saccharide in the composition conjugated to diphtheria toxoid (DT) or CRM197.

Description

POLYAC AIR CONJUGATE VACCINE PNEUMOCOCCAL Field of the Invention The present invention relates to a Streptococcus pneumoniae vaccine. Background of the Invention Children under 2 years of age do not produce an immune response to most polysaccharide vaccines, therefore it has been necessary to produce immunogenic polysaccharides by chemical conjugation to a protein carrier.

Coupling the polysaccharide, a T-independent antigen, to a protein, a T-dependent antigen, confers on the polysaccharide the T-dependence characteristics including isotype switching, affinity maturation, and memory induction. However, there may be problems with repeated administration of polysaccharide-protein conjugates, or with the combination of polysaccharide-protein conjugates to form polyvalent vaccines. For example, it has been reported that a polysaccharide vaccine (PRP) of Hemophilus influenzae type b using tetanus toxoid (TT) as a protein carrier was tested in a dosing interval with simultaneous immunization with TT (free) and a pneumococcal polysaccharide-TT conjugate vaccine following a standard children's program. Although the dosage of pneumococcal vaccine was increased, the immune response decreased to the PRP polysaccharide portion of the Hib conjugate vaccine, indicating the immunological interference of the polysaccharide, possibly via the use of the same carrier protein (Dagan et al., Infect. Immun. (1998); 66: 2093-2098). The effect of the carrier-protein dosage on the humoral response to the protein by itself has also been shown to be multifaceted. In human children it was reported that increasing the dosage of a tetravalent tetanus toxoid conjugate resulted in a decreased response to the tetanus carrier (Dagan et al., Supra). The classical analysis of these effects of combination vaccines has been described as an epitope suppression induced by the carrier, which is not fully understood, but has been believed to result from an excessive amount of a carrier protein (Fattom, Vaccine 17: 126 (1999)). This seems to result in the competition of Th cells, by B cells with the carrier protein, and B cells with the polysaccharide. If B cells predominate on the carrier protein, there are not enough Th cells available to provide the necessary help for the B cells specific for the polysaccharide. However, the observed immunological effects have been inconsistent, with the total amount of carrier protein in some cases the immune response is increased, and in other cases the immune response is decreased. Therefore the technical difficulty remains multiple polysaccharide conjugates to a single effective vaccine formulation. Streptococcus pneumoniae is a gram-positive bacterium responsible for considerable morbidity and mortality (particularly in young and old people), causing invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonization, such as acute Otitis media. The rate of pneumococcal pneumonia in the United States of America for people over 60 years of age is estimated to be 3 to 8 per 100,000 in 20% of cases, this leads to bacteremia, and other manifestations such as meningitis, with a mortality rate of approximately 30% even with antibiotic treatment. The pneumococcus is encapsulated with a chemically bound polysaccharide that confers serotype specificity. There are 90 known serotypes of pneumococci, and the capsule is the main virulent determinant for pneumococci, since the capsule not only protects the internal surface of the bacteria against complement, but by itself is poorly immunogenetically. Polysaccharides are antigens independent of T, and can not be processed or presented in MHC molecules to interact with T cells. However, they can stimulate the immune system through an alternative mechanism that involves the cross-linking of surface receptors in cells. of B.

It was shown in several experiments that the protection against invasive pneumococcal disease is very strongly correlated to the antibody specific for the capsule, and the protection is serotype specific. Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children. Likewise, the poor responses of the elderly population to pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol 42: 265-270], therefore causes the increased incidence of bacterial pneumonia in this population [Verghese and Berk, (1983) Medicine (Baltimore) 62 271-285]. The main clinical syndromes caused by S. pneumoniae are recognized and widely discussed in all standard medical textbooks (Fedson DS, Muscher DM.

Plotkin SA, Orenstein WA, editors. Vaccines 4rth 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 other normally sterile site (Musher DM, Streptococcus pneumoniae In Mandell GL, Bennett JE, Dolin R (eds). Principies and Practice of Infectious diseases (5th ed). New York, Churchill Livingstone, 2001, p2128-2147). It is recognized that chronic obstructive pulmonary disease (COPD) comprises several conditions (obstruction of air circulation, chronic bronchitis, bronchiolitis or small airway disease and emphysema) that frequently coexist. Patients suffer exacerbations of their condition that are usually associated with increasing dyspnea, and cough that can be productive of mucus or purulent sputum have frequently increased (Wilson, Eur Respir J 2001 17: 995-1007). COPD is defined physiologically by the presence of irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease.

Thoracic Society. Am J Respir Crit Care Med. 1995 Nov; 152 (5 Pt 2): S77-121). Exacerbations of COPD are frequently caused by bacterial infection (eg, pneumococcal) (Sethi S, Murphy TF, Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review.

Clin Mícrobiol Rev. April 2001; 14 (2): 336-63). Thus it is an object of the present invention to develop an improved formulation of a polysaccharide conjugate vaccine of multiple serotype Streptococcus pneumoniae. Brief Description of the Figures Figure 1 is a bar graph showing the immunogenicity of the 11-valent conjugate in Rhesus monkeys. The smaller bars represent the GMC after two inoculations with the 11-valent conjugate in the aluminum phosphate adjuvant. The darker bars represent the GMC after two inoculations with the 11-valent conjugate in adjuvant C. Figure 2 is a bar graph showing memory B cells for PS3 after inoculation with the 11-valent conjugate in adjuvant C or aluminum phosphate adjuvant. Figure 3 is a bar graph showing the immunogenicity of anti-polysaccharide 19F in Balb / C mice for 4 single 4-valent polysaccharides and 4-valent dPly conjugates. Figure 4 is a bar graph showing the immunogenicity of anti-polysaccharide 22F in Balb / C mice for 4-valent simple polysaccharides and 4-valent PhtD conjugates. Figure 5 is a bar graph showing the response to anti-22F IgG in Balb / c mice. Figure 6 is a bar graph showing the anti-22F opsonophagocytosis titers in Balb / c mice. Figure 7 is a bar graph comparing IgG responses induced in young C57B1 mice after immunization with the 13-valent conjugate vaccine formulated in different adjuvants. Figure 8 is a bar graph showing the protective efficacy of different vaccine combinations in a mono pneumonia model.

Figure 9 is a bar graph showing the anti-PhtD IgG response in Balb / c mice after immunization with 22F-PhtD or 22F-AH-PhtD conjugates. Figure 10 shows the protection against the challenge with the type 4 pneumococcus in mice after immunization with 22F-PhtD or 22F-AH-PhtD. Description of the Invention The present invention provides an improved Streptococcus pneumoniae vaccine comprising 10 or more io (for example 11, 12, 13, 14, or 15 or more) capsular saccharides of different serotypes of S. pneumoniae conjugated to 2 or more carrier proteins, wherein the vaccine comprises the capsular saccharide of serotype 19F conjugated to diphtheria toxoid or CRM197, and wherein the vaccine optionally comprises additionally the protein D of Hemophilus influenzae as a free protein or as another carrier protein or both. For the purposes of this invention, "immunize a human host against COPD exacerbations" or "treat or prevent exacerbations of COPD" or "reduce the severity of exacerbations of COPD "refers to a reduction in the incidence or rate of COPD exacerbations (for example a reduction in the index of 0.1, 0.5, 1, 2, 5, 10, 20% or more) or a reduction in the severity of COPD exacerbations as defined above, for example within a group of patients immunized with the compositions or vaccines of the invention. Commonly the Streptococcus pneumoniae vaccine of the present invention will comprise capsular saccharide antigens (preferably conjugates), wherein the saccharides are derived from at least ten serotypes of S. pneumoniae. The number of capsular saccharides of S. pneumoniae can range from 10 different serotypes (or "V", valences) 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 comprise conjugated saccharides of S. pneumoniae and unconjugated saccharides of S. pneumoniae. Preferably, the total number of serotypes of the saccharide is less than or equal to 23. For example, the invention may comprise 10 conjugated serotypes and 13 unconjugated saccharides. In a similar manner, the vaccine may comprise 11, 12, 13, 14, 15 or 16 conjugated saccharides and 12, 11, 10, 9, 8 or 7, respectively, unconjugated saccharides. In one embodiment the polyvalent pneumococcal vaccine of the invention will be selected from the following serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, although it is appreciated that one or two of other serotypes can be substituted depending on the age of the recipient receiving the vaccine and the geographical location where the vaccine will be administered, for example the serotypes 6A can be included in the list. For example, a 10-valent vaccine it can comprise the polysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. The 11-valent vaccine may also include serotype 3 serotypes. A 12- or 13-valent pediatric (infant) vaccine may also include a 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 vaccine for 13-valent 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 22F1 or 12F and 15B, or 12F and 19A, or 12F and 22F, or 15B and 19A, or 15B and 22F. A pediatric 14-valent 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 the capsular saccharides derived from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (preferably conjugated). In another embodiment of the invention, at least 11 saccharide antigens (preferably conjugates) are included, for example, capsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. In a further embodiment of the invention, at least 12 or 13 saccharide antigens are included, example, a vaccine may comprise 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 (for example 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 comprise the protein (PD) of Hemophilus influenzae (see for example EP 0594610). Hemophilus influenzae is an important organism causing otitis media, and the present inventors have shown that including this protein in a Streptococcus pneumoniae vaccine will provide a level of protection against otitis media related to Hemophilus influenzae (reference POET publication). In one embodiment, the vaccine composition comprises protein D. In one aspect, PD is present as a carrier protein for one or more of the saccharides. In another aspect, protein D could be present in the vaccine composition as a free protein. In a further aspect, protein D is present as a carrier protein and as a free protein. Protein D can be used as an integral 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 saccharides can be conjugated to protein D. In this aspect, protein D can also be present as a free protein. The vaccine of the present invention comprises two or more different types of carrier protein. Each type of carrier protein can act as a carrier for more than one saccharide, such saccharides can be the same or different. For example, serotypes 3 and 4 can be conjugated to the same carrier protein, to the same carrier protein molecule or to different molecules of the same carrier protein. In one embodiment, two or more different saccharides can be conjugated to the same carrier protein, to the same carrier protein molecule or to different molecules of the same carrier protein. Each capsular saccharide of Streptococcus pneumoniae can be conjugated to a carrier protein selected independently of the group consisting of TT, DT, CRM197, fragment C of TT, fusions of PhtD, PhtDE (particularly those described in WO 01/98334 and WO 03/54007 ), detoxified pneumolysin and protein D, other than the serotype saccharide 19F which is always conjugated to DT or CRM 197, preferably DT. A more complete list of protein carriers that can be used in the conjugates of the invention is presented below. If the protein carrier is the same for 2 or more saccharides in the composition, the saccharides could be conjugated to the same molecule of the protein carrier (carrier molecules having 2 or more different saccharides conjugated thereto) [see for example WO 04/083251]. Alternatively the saccharides each can be conjugated separately to different molecules of the protein carrier (each molecule of protein carrier only has one type of saccharide conjugated thereto). The carrier protein conjugated to one or more of the capsular saccharides of S. pneumoniae in the io conjugates present in the immunogenetic compositions of the invention is optionally a member of the proteins of the polyhistidine triad (Pht) family, fragments or fusion proteins of it. The PhtA, PhtB, PhtD or PhtE proteins can have an amino acid sequence that share an i5 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with a sequence described in WO 00 / 37105 or WO 00/39299 (for example with the 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 entirely or partially by 2, 3 or 4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins are PhtA / B, PhA / D, PhtA / E, PhtB / A, PhtB / D, PhtB / E PhtD / A PhtD / B, PhtD / E, PhtE / A, PhtE / B and PhtE / D, where proteins they join the first one mentioned in terminal N (see for example WO01 / 98334). 25 Where fragments of Pht proteins are used (separately or as part of a fusion protein), each fragment optionally contains one or more triad histidine motifs and / or coiled spiral regions of such polypeptides. A triad motif of histidine is the polypeptide portion having the sequence HxxHxH, where H is histidine and x is an amino acid other than histidine. A coiled spiral type region is a region provided by the algorithm "Coils", Lupus, A et al. (1991) Science 252, 1162-1 164. In one embodiment each fragment includes one or more triad histidine motifs as well as at least one coiled spiral type region. In one embodiment each fragment contains exactly or at least 2, 3, 4 or 5 triad histidine motifs (optionally, with the native Pht sequence between 2 or more triads or intra-triads that is more than 50, 60, 70 , 80, 90 or 100% identical to a native pneumococcal intra-triad Pht sequence - for example the intra-triad sequence shown in SEQ ID NO: 4 of WO 00/37105 for PhtD). In one embodiment each fragment contains exactly or at least 2, 3 or 4 coiled spiral regions. In one embodiment a Pht protein described herein includes the integral protein with the linked signal sequence, the mature integral protein with the signal peptide removed (for example 20 amino acids at the N-terminus), natural variants of the Pht protein and immunogenetic fragments of the Pht protein (for example fragments as described above or polypeptides comprising at least 15 or 20 contiguous amino acids of an amino acid sequence in WO00 / 37105 or WO00 / 39299, wherein the polypeptide is capable of producing an immune response specific for the amino acid sequence in WO00 / 37105 or WO00 / 39299) Particularly, the term "PhtD as used herein includes the integral protein with the bound signal sequence, the mature integral protein with the signal peptide eliminated (for example 20 amino acids at the N-terminus), natural variants of PhtD and fragments. PhtD immunogenetics (e.g. fragments as described above or polypeptides comprising at least 15 or 20 contiguous amino acids of a PhtD amino acid sequence in WO00 / 37105 or WO00 / 39299, wherein the polypeptide is capable of producing a specific immune response for the amino acid sequence of PhtD in WOOO / 37105 or WO00 / 39299 (for example SEQ ID NO: 4 of WO 00/37105 for PhtD). of protein is the same for 2 or more saccharides in the composition, the saccharides could be conjugated to the same molecule of the protein carrier (carrier molecules having 2 or more different saccharides conjugated thereto) [see for example WO 04/083251]. Alternatively the saccharides can each be separately conjugated to different protein carrier molecules (each molecule of the protein carrier only has one type of saccharide conjugated thereto).

Examples of carrier proteins that can be used in the present invention are DT (diphtheria toxoid), TT (tetanus toxide) or fragment C of TT, DT CRM197 (a mutant of DT), other mutants of DT point, 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 to Gly and other mutations described in US 4709017 or US 4950740; the 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 the fragment described in US 5843711, pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63, 2706-13) including ply detoxified in a certain manner for example dPLY-GMBS (WO 04081515, PCT / EP2005 / 010258) or dPLY-formalin, PhtX, including the PhtA, PhtB, PhtD, PhtE and PhtDE fusions of the Pht proteins for example PhtBE fusions (WO 01/98334 and WO 03/54007), (Pht AE is described in more detail below) , OMPC (meningococcal outer membrane protein - generally extracted from serogroup B of N. meningitidis - EP0372501), PorB (N. meningitidis), PD (protein D from Hemophilus influenzae - see, for example, EP 0 594 610 B), or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP0427347), proteins of thermal shock (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins comprising multiple cell epitopes Human CD4 + T of several antigens derived from pathogens (Falugi et al (2001) Eur J Immunol 31; 3816-3824) such as the N19 protein (Baraldoi et al (2004) Infect Immun 72; 4884-7), protein superficial pneumococcal PspA (WO 02/091998), iron absorption proteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761). Nurkka et al. Pediatric Infectious Disease Journal. 23 (11): 1008-14, 2004 Nov. describes an 11-valent pneumococcal vaccine with all serotypes conjugated to PD. However, the present inventors have shown that opsonophagocytic activity was improved for antibodies induced with conjugates having 19F conjugated to DT compared to 19F conjugated to PD. In addition, the present inventors have shown that a greater cross-reactivity to 19A is observed with 19F conjugated to DT. It is therefore a feature of the composition of the present invention that serotype 19F is conjugated to DT or CRM 197. In one aspect, serotype 19F is conjugated to DT. The remaining serotypes of the saccharide of the immunogenic composition can all be conjugated to one or more carrier proteins other than DT (ie only 19F is conjugated to DT), or they can be divided among one or more carrier proteins that are not DT and DT by itself. In one modality, 19F is conjugated to DT or CRM 197 and all the remaining serotypes are conjugated to PD. In another modality, 19F is conjugated to DT or CRM 197, and the remaining serotypes are divided between PD, and TT or DT or CRM 197. In an additional modality, 19F is conjugated to the saccharide of DT or CRM 197 and not more than one conjugates to TT. In one aspect of this embodiment, the saccharide is 18C or 12F. In another embodiment, 19F is conjugated to the saccharides of DT or CRM 197 and no more than two are conjugated to TT. In another modality, 19F is conjugated to DT or CRM 197, and the remaining serotypes are divided between PD, TT and DT or CRM 197. In another modality, 19F is conjugated to DT or CRM 197, and the remaining serotypes are divided among PD , TT and pneumolysin. In another modality, 19F is conjugated to DT or CRM 197, and the remaining serotypes are divided between PD, TT and CRM 197. In another modality, 19F is conjugated to DT or CRM197 and the remaining serotypes are divided among PD, TT, pneumolysin and optionally the fusion protein of PhtD or PhtD / E. In another embodiment, 19F is conjugated to DT or CRM197, 19A is conjugated to pneumolysin or TT, one (two or three) additional saccharide is conjugated to TT, an additional saccharide is conjugated to PhtD or PhtD / E and all the other saccharides are they conjugate PD. In a further modality 19F is conjugated to DT or CRM197, 19A is conjugated to pneumolysin, one (two or three) additional saccharide is conjugated to TT, an additional saccharide is conjugated to pneumolysin, 2 or more saccharides are conjugated to PhtD or PhtD / E and all other saccharides are conjugated to PD. In one embodiment, the immunogenetic composition of the invention comprises protein D of Hemophilus influenzae. Within this modality, if the PD is not one of the carrier proteins used to conjugate any saccharide other than 19F, for example 19F is conjugated to DT while the other serotypes are conjugated to one or more different carrier proteins other than PD, then PD will be present in the vaccine composition as a free protein. If PD is one of the carrier proteins used to conjugate saccharides other than 19F, then PD may optionally be present in the vaccine composition as a free protein. The term "saccharide" throughout this specification may indicate the polysaccharide or oligosaccharide and includes both. The polysaccharides are isolated from the bacteria and can be sized to a certain degree by known methods (see for example EP497524 and EP497525) and preferably by microfluidization. The polysaccharides can be sized to reduce the viscosity in polysaccharide samples and / or to improve the filterability of the conjugated products. Oligosaccharides have a low number of repeat units (commonly 5-30 repeat units) and are commonly hydrolyzed polysaccharides.

The capsular polysaccharides of Streptococcus pneumoniae comprise repeated oligosaccharide units which may contain up to 8 sugar residues. For a review of the Oligosaccharide units for the serotype of Streptococcus pneumoniae essential see JONES, Christopher. Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An. Acad. Bras. Cieñe, June 2005, vol.77, no.2, p.293-324. ISSN 0001-3765. In one embodiment, a capsular saccharide antigen can be an integral polysaccharide, however in others it can be an oligosaccharide unit, or a saccharide chain of shorter length than the native of the repeating oligosaccharide units. In one embodiment, all saccharides present in the vaccine are polysaccharides. The integral polysaccharides can be "dimensioned" ie their size can be reduced by several methods such as acid hydrolysis treatment, hydrogen peroxide treatment, sizing by Emulsiflex® followed by a hydrogen peroxide treatment to generate the fragments of oligosaccharide or microfluidization. The inventors have also observed that the aim of the technique has been to use the oligosaccharides to facilitate the production of conjugates. The inventors have found that by using the native or slightly sized polysaccharide conjugates, one or more of the following advantages can be obtained: 1) a conjugate having high immunogenicity that is filterable, 2) the ratio of polysaccharide to protein in the conjugate is can alter such that the ratio of polysaccharide to protein (w / w) in the conjugate can be increased (which can have an effect on the effect of deletion of the carrier), 3) the immunogenetic conjugates prone to hydrolysis can be stabilized by the use of larger saccharides for conjugation. The use of larger polysaccharides can result in greater cross-linking with the conjugate carrier and can decrease the release of free saccharide from the conjugate. The conjugate vaccines described in the prior art tend to depolymerize the polysaccharides before conjugation to improve conjugation. The present inventors have found that saccharide conjugate vaccines that maintain a larger saccharide size can provide a good immune response against the disease. pneumococcal The immunogenetic composition of the invention may thus comprise one or more saccharide conjugates wherein the average size (e.g., weight average molecular weight; Mw) of each saccharide before conjugation is above 80 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa or 1000 kDa. In one embodiment one or more saccharide conjugates of the invention must have an average pre-conjugate size of saccharide of 50-1600, 80-1400, 100-1000, 150-500, or 200-400 kDa (Note that where the average size is Mw, the "kDa" units must be replaced in the present by "x103"). In a modality the conjugate after the conjugation must be easily filterable through a 0.2 micron filter such that a production of more than 50, 60, 70, 80, 90 or 95% is obtained after the filtration compared with the sample before filtration. For the purposes of the invention, the "native polysaccharide" refers to a saccharide that has not been subjected to a process (eg post-purification), the purpose of which is to reduce the size of the saccharide. A polysaccharide can be slightly reduced in size during normal purification procedures. Such a saccharide remains natural. Only if the polysaccharide has been subjected to the dimensioning techniques the polysaccharide would not be considered native. For the purposes of the invention, "sized by a factor of up to x2" means that the saccharide is subjected to a process intended to reduce the size of the saccharide but to retain a size more than half the size of the native polysaccharide. X3, x4 etc. they must be interpreted in the same way, that is, the saccharide is subjected to a process designed to reduce the size of the polysaccharide but to keep a size greater than a third, fourth etc. of the size of the native polysaccharide.

In one aspect of the invention, the immunogenic composition comprises 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 of the saccharides of S. pneumoniae is a native polysaccharide. In one aspect of the invention, the composition immunogenetics comprises the saccharides of Streptococcus pneumoniae from at least 10 serotypes conjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or each of the saccharides 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 the average molecular weight (or size) of a saccharide herein refers to the weight average molecular weight of the saccharide measured before conjugation and is measured by MALLS. The MALLS technique is well known in the art and is commonly performed as described in example 2. For the MALLS analysis of pneumococcal saccharides, two columns (TSKG6000 and 5000PWxl) can be used in the combination and the saccharides are eluted in water. The saccharides are detected using a light scattering detector (for example Wyatt Dawn DSP equipped with an argon laser of 10 mW at 488 nm) and a inferometric refractometer (for example Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498 nm). . In one embodiment the saccharides of S. pneumoniae are the native polysaccharides or native polysaccharides that have been reduced in size during a normal extraction process. In one embodiment, the saccharides of S. pneumoniae are dimensioned by mechanical division, for example by microfluidization or sonic treatment. Microfluidization and sonic treatment have the advantage of decreasing the size of the larger native polysaccharides sufficiently to provide a filterable conjugate. The resignation is by a factor not greater than x20, x10, x8, x6, x5, x4, x3 or x2. In one embodiment, the immunogenetic composition comprises the conjugates of S. pneumoniae that are made from a mixture of native polysaccharides and saccharides that are sized by a factor not greater than x20. In one aspect of this embodiment, most 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 via a linkage, for example a bifunctional linkage. The linkage is optionally heterobifunctional or homobifunctional, having for example a reactive amino group and a reactive carboxylic acid group, 2 reactive amine groups or two reactive carboxylic acid groups. The bond has, for example, between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possible link is ADH. Other linkages 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 link for the conjugation saccharide of serotype 18C. In one embodiment, ADH is used as a link for the conjugation saccharide of serotype 22F. The saccharide conjugates present in the immunogenetic compositions of the invention can be prepared by any known coupling technique. The conjugation method can be based on the activation of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide can thus be coupled directly or via a spacer group (linkage) to an amino group on the carrier protein. For example, the spacer could be cystamine or cysteamine to give a thiolated polysaccharide that could be coupled to the carrier via 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 (e.g. EDAC or EDC) via a carboxyl group in the protein carrier. Such conjugates are described in PCT Published Application WO 93/15760 of Uniformed Services University and WO 95/08348 and WO 96/29094. Other convenient techniques use the carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO98 / 42721. The conjugation may involve a carbonyl bond that can be formed by the reaction of a free hydroxyl group of the saccharide with CD1 (Bethell et al J. Biol. Chem. 1979, 254; 2572-4, Hearn et al., J. Chromatogr 1981. 218; 509-18) followed by the reaction of a protein to form a carbamate linkage. This may involve the reduction of the anomeric term to a primary hydroxyl group, the optional protection / deprotection of the reaction of the primary hydroxyl group with CDl to form a CD1 carbamate intermediate and coupling the CD1 carbamate intermediate with an amino group in an protein. The conjugates can also be prepared by direct reducing 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. An additional method involves the coupling of an activated saccharide of cyanogen bromide (or CDAP) derivative with adipic acid dihydrazide (ADH) to the protein carrier by the carbodiimide condensation (Chu C et al Infect Immunity, 1983 245256), for example using EDAC. In one embodiment, a hydroxyl group (preferably a activated hydroxyl group for example an activated hydroxyl group to make a cyanate ester [for example with CDAP]) in a saccharide, binds to an amino or carboxylic group in a protein directly or indirectly (via a bond). Where a bond is present, a hydroxyl group in a saccharide is preferably linked to an amino group in a bond, for example using the conjugation of CDAP. An additional amino group in the linkage, for example ADH, can be further conjugated to a carboxylic acid group in a protein, for example using carbodiimide chemistry, for example using EDAC. In one embodiment, the pneumococcal capsular polysaccharides are conjugated to the first link before the linkage is conjugated to the carrier protein. Alternatively the linkage can be conjugated to the carrier prior to conjugation to the saccharide. A combination of techniques can also be used, with some saccharide-protein conjugates that are prepared by CDAP, and some by reductive amination. In general the following types of chemical groups in a protein carrier can be used for coupling / conjugation. A) Carboxyl (for example via aspartic acid or glutamic acid). In one embodiment this group binds to the amine groups in the saccharides directly or to an amino group in a bond with the carbodiimide chemistry for example with EDAC. B) Amino group (for example via lysine). In a This group is linked to the carboxyl groups in the saccharides directly or to a carboxyl group in a bond with the carbodiimide chemistry for example with EDAC. In another embodiment this group binds to the hydroxyl groups activated with CDAP or CNBr in the saccharides directly or to such groups in a bond; to saccharides or linkages having an aldehyde group; to saccharides or bonds having a succinimide ester group. C) S u If h id ri lo (for example via cysteine). In one embodiment this group is linked to an acetylated saccharide of bromine or chlorine or a bond with maleimide chemistry. In one embodiment, this group is activated / modified with bis-diazobenzidine. D) Hydroxyl group (for example via tyrosine). In one embodiment, this group is activated / modified with bis-diazobenzidine. E) Imidazolyl group (for example via histidine). In one embodiment, this group is activated / modified with bis-diazobenzidine. F) Guanidyl group (for example via arginine). G) Indolyl group (for example via tryptophan). In a saccharide, in general the following groups can be used for a coupling: OH, COOH or NH2. The aldehyde groups can be generated after different treatments known in the art for example: periodate, hydrolysis of acid, hydrogen peroxide, etc. Direct coupling methods Saccharide-OH + CNBr or CDAP? cyanate ester + NH2-Prot ? conjugate saccharide-aldehyde + NH2-Prot? Schiff base + NaCNBH3 - > conjugate saccharide-COOH + NH2-Prot + EDAC? conjugate saccharide-NH2 + COOH-Prot + EDAC -? conjugate Methods of indirect coupling via a separator (link) Saccharide-OH + CNBr or CDAP? cyanate ester + MH2 NH2? saccharide NH2 + COOH-Prot + EDAC > conjugate Saccharide-OH + CNBr or CDAP -? cyanate ester + NH2 SH -? saccharide SH + sH-Prot (native protein with a cysteine exposed or obtained after modification of the amino groups of 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? conjugate Saccharide-COOH + EDAC + NH2 --- NH2? saccharide NH2 + EDAC + COOH-Prot -? conjugate Saccharide-COOH + EDAC + NH2 SH? saccharide SH + sH-Prot (native 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 + NH2 SH? saccharide SH + maleimide-Prot (modification of amino groups)? conjugate Saccharide-COOH + EDAC + NH2 ---- SH? Saccharide-SH + haloacetylated-Prot? conjugate Saccharide-Aldehyde + NH2 NH2 -? saccharide NH2 + EDAC + COOH-Prot? Conjugate Note: Instead of EDAC above, any suitable carbodiimide can be used. In conclusion, the types of chemical groups of protein carrier that can generally be used to couple with a saccharide, are amino groups (for example in lysine residues), COOH groups (for example in aspartic and glutamic acid residues) and groups SH (if accessible) (for example in cysteine residues). Preferably the ratio of the 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 to saccharide protein 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 via a link using CDAP and EDAC. For example, 18C or 22F can be conjugated to a protein via a bond (for example those with two hydrazino groups at their ends such as ADH) using CDAP and EDAC as described above. When it's used a link, CDAP can be used to conjugate the saccharide to a link and EDAC can then be used to conjugate the link to a protein or, alternatively EDAC can be used first to conjugate the link to the protein, after which CDAP can be used. use to conjugate the link to the saccharide. Generally the immunogenetic composition of the invention may comprise a dose of each saccharide conjugate between 0.1 and 20 μg, 1 and 10 μg or 1 and 3 μg of saccharide. In one embodiment, the immunogenetic composition of the invention contains each capsular saccharide of S. pneumoniae in a dose of between 0.1-20 μg; 0.5-10 μg; 0.5-5μg or 1-3 μg of saccharide. In one embodiment, the capsular saccharides may be present in different dosages, for example some capsular saccharides may be present in a dose of exactly 1 μg or some capsular saccharides may be present in a dose of exactly 3 μg. In one embodiment, the saccharides of serotypes 3, 18C and 19F (or 4, 18C and 19F) are present in a higher dose than other saccharides. In one aspect of this embodiment, serotypes 3, 18C and 19F (or 4, 18C and 19F) are present at a dose of approximately or exactly 3 μg while other saccharides in the immunogenic composition are present at a dose of approximately or exactly 1 μg. "Around" or "approximately" are defined as within 10% more or less of the given figure for the purposes of the invention. In one embodiment, at least one of the capsular saccharides of S. pneumoniae is conjugated directly to a carrier protein (for example using one of the chemistries described above). Preferably 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 directly linked to the carrier protein by CDAP (see WO 95/08348 and WO 96/29094). The immunogenetic composition may comprise Streptococcus pneumoniae proteins, in the present so-called Streptococcus pneumoniae proteins of the invention, such 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 any exposed surface, at least during the part of the life cycle of the pneumococcus, or are the proteins that are secreted or released by 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 (where X is any amino acid, eg, the polyhistidine (PhtX) triad family), proteins of choline binding, proteins that have a type I signal sequence motif (for example, Sp101), proteins having a motif LPXTG (where X is any amino acid, eg, Sp128, Sp130), and toxins (eg, Ply) Preferred examples within these categories (or motifs) are the following proteins, or immunologically functional equivalents thereof In one embodiment, the immunogenetic composition of the invention comprises at least 1 protein selected from the group consisting of the family of triad of histidine (PhtX), family of choline binding protein (CbpX) , Truncated CbpX truncated, LytX family, truncated LytX, Truncated LytX chimeric proteins from CbpX (or fusions), Pneumolysin (Ply), PspA, PsaA, Sp128, S1101, Sp130, Sp125 and Sp133. Further, the immunogenetic composition comprises 2 or more proteins selected from the group consisting of the family of histidine po triads (PhtX), choline-binding protein family (CbpX), truncated CbpX, LytX family, truncated d e LytX, truncated LytX-truncated chimeric proteins of CbpX (or fusions), pneumolysin (Ply), PspA, and Sp128 In a further embodiment, the immunogenetic composition comprises 2 or more proteins selected from the group consisting of the triad family of poly histi ina (PhtX), family of choline-binding protein (CbpX), truncated CbpX, LytX family, truncated LytX, truncated chimeric proteins of LytX-truncated CbpX (or fusions) pneumolysin (Ply), and Sp128 Pht family (triad of pohhistidina) comprises the 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) coiled-spiral regions, one N terminal conserved and a heterogeneous C terminal. It is present in all strains of pneumococci tested. The homologous proteins have also been found in the other Streptococcis and Neisseria. In one embodiment of the invention, the Pht protein of the invention is PhtD. It is understood, however, that the terms Pht A, B, D, and E refer to the proteins having sequences described in the later citations as well as natural (and artificial) variants thereof having a sequence homology that is of at least 90% identical to the proteins referred to. Preferably it is at least 95% identical and more preferably is 97% identical. With respect to PhtX proteins, PhtA is described in WO 98/18930, and is also referred to as Sp36. As noted above, it is a protein of the polyhistidine triad family and has the type II signal motif of LXXC. PhtD is described in WO 00/37105, and is also referred to as SpO36D. As noted above, it is also a protein of the polyhistidine triad family and has the type II signal motif of LXXC. PhtB is described in WO 00/37105, and is also referred to as SpO36B. Another member of the PhtB family is the C3 degradation polypeptide, as described in WO 00/17370. This protein is also from the polyhistidine triad family and has the type II signal motif of LXXC. A preferred immunologically functional equivalent is the Sp42 protein described in WO 98/18930. A truncated PhtB (approximately 79kD) is described in WO99 / 15675 which is also considered a member of the PhtX family. PhtE is described in WO00 / 30299 and is referred to as BVH-3. Where any Pht protein is referred to herein, it is understood that the 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 to PhtB is also a reference to fusions of PhtDE or PhtBE as found, for example, in WO0198334. Pneumolysin is a multi-functional toxin with distinct cytolytic (hemolytic) and complement activation activities (Rubins et al., Am. Res. Cit Care Med, 153: 1339-1346 (1996)). The toxin is not secreted by pneumococci, but is released during the lysis of pneumococci under the innce of autolysin. Its effects include, for example, the stimulation of the production of inflammatory cytokines by human monocytes, the inhibition of the effect of cilia on the human respiratory epithelium, and the decrease of bactericidal activity and migration of neutrophils. The most obvious effect of pneumolysin It is in the lysis of red blood cells, which involves binding to cholesterol. Because it is a toxin, it needs to be detoxified (ie, non-toxic to a human when provided at a convenient dosage for protection) before it can be administered in vivo. The expression and cloning of wild or native pneumolysin is known in the art. See, for example, Walker et al. (Infec Immun, 55-1184-1189 (1987)), Mitchell et al. (Biochim Biophys Acta, 1007: 67-72 (1989) and Mitchell et al (NAR, 18: 4010 (1990)). Ply detoxification can be conducted by chemical means, for example by undergoing formalin or glutaraldehyde treatment or a combination of both (WO 04081515, PCT / EP2005 / 010258), such methods are well known in the art for various toxins Alternatively, Ply can be genetically detoxified Thus, the invention comprises the derivatives of pneumococcal proteins which can be for example, mutated proteins The term "mutated" is used herein to refer to 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 method For example, as described above, a ply mutant protein can be altered to be biologically inactive while still maintaining its immunogenic epitopes, see, for example, WO90 / 06951, Berry et al. (Infecí Immun, 67 981-985 (1999)) and WO99 / 03884.

As used herein, it is understood that the term "Ply" refers to mutated or detoxified pneumolysin suitable for medical (ie, non-toxic) use. In reference to 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 choline binding proteins are non-covalently bound to the phosphorylcholine portions of cell wall teichoic acid and lipoteichoic acid associated with the membrane. Structurally, they have several regions in common with respect to the entire family, although the exact nature of the proteins (amino acid sequence, length, etc.) may vary. Generally choline binding proteins comprise an N (n) terminal region, conserved repeat regions (R1 and / or R2), a proline (p) rich region and a conserved choline binding region (c), are composed of multiple repeats, comprising approximately one half of the protein. As used in this application, the term "choline binding protein family (CbpX)" is selected from the group consisting of choline binding proteins as identified in WO97 / 41151, PbcA, SpsA, PspC, CbpA, CbpD, and CbpG. CbpA is described in WO97 / 41151. CbpD and CbpG are described in WO00 / 29434. PspC is described in WO97 / 09994. PbcA is described in WO98 / 21337. SpsA is a binding protein of hill 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 where "CbpX" is defined above and "truncated" refers to CbpX proteins that lack 50% or more of the choline binding region (c). Preferably such proteins lack the entire binding region of whole choline. More preferably, such a truncated protein lacks (i) the choline binding region and (ii) a portion of the N-terminal half of the protein as well as still maintains at least one repeat region (R1 or R2). Even more preferably, the truncated ones have 2 repeating regions (R1 and R2). Examples of such preferred embodiments are NR1xR2 and R1xR2 as illustrated in WO99 / 51266 or WO99 / 51188, however, other choline binding proteins lacking a similar choline binding region are also contemplated within the scope of this invention. . The LytX family are membrane associated proteins associated with cell lysis. The N-terminal domain comprises the choline binding domains, however the LytX family does not have all the characteristics found in the CbpA family known above and thus for the present invention, the LytX family is considered distinct from the CbpX family. In contrast to the CbpX family, the C-terminal domain contains the catalytic domain of the LytX protein family. The family includes LytA, B and C. With 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 LytX truncates where "LytX" is defined above and "truncated" refers to LytX proteins that lack 50% or more of the choline binding region. Preferably such proteins lack the entire choline binding region. Still a preferred additional embodiment of this invention are the truncated LytX-truncated CbpX chimeric proteins (or fusions). Preferably they comprise NR1xR2 (or R1xR2) of CbpX and the C-terminal portion (Cterm, ie, lack the choline binding domains) of LytX (for example LytCCterm or Sp91Cterm). Preferably CbpX is selected from the group consisting of CbpA, PbcA, SpsA and PspC. Even more preferably, it is CbpA. Preferably, LytX is LytC (also referred to as Sp91). Another embodiment of the present invention is a PspA or truncated PsaA that lack the choline binding domain (c) and are expressed as the fusion protein with LytX. Preferably, LytX is LytC. With respect to PsaA and PspA, both are known in the art. For example, PsaA and the transmembrane deletion variants thereof have been described by Berry and Patón, Infect Immun 1996 Dec; 64 (12): 5255-62. PspA and the transmembrane deletion variants thereof have been described in, for example, US 5804193, WO 92/14488, and WO 99/53940. Sp128 and Sp130 are described in WO00 / 76540. Sp125 is an example of a pneumococcal surface protein with the cell wall anchored motif of LPXTG (where X is any amino acid). Any protein within this class of pneumococcal surface protein with this motif has been found useful in the context of this invention, and is therefore considered another protein of the invention. Sp125 by 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 # y85993). It is characterized by a type I signal sequence. SP 133 is described in WO 98/06734 (where it has the reference # y85992). It is also characterized by a type I signal sequence. Examples of preferred Moraxella catarrhalis protein antigens that can be included in a combination vaccine (especially for the prevention of otitis media) are: OMP106 [WO 97/41731 (Antex) 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) Infect Immun. 61.2003-2010]; UspA1 and / or UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR (PCT / EP99 / 03824), PilQ (PCT / EP99 / 03823); OMP85 (PCT / EPOO / 01468); Iipo06 (GB 9917977 2); Iipo10 (GB 9918208.1), lipo11 (GB 9918302 2), lipo18 (GB 9918038.2), P6 (PCT / EP99 / 03038); D15 (PCT / EP99 / 03822); OmplAI (PCT / EP99 / 06781); Hly3 (PCT / EP99 / 03257); and OmpE. Examples of non-type Hemophilus influenzae antigens or fragments thereof that may be included in a combination vaccine (especially for the prevention of otitis media) include fimbrin protein [(US 5766608 - Ohio State Research Foundation)] and fusions comprising peptides thereof [eg peptide fusions of 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 understood that the immunogenetic composition comprises all the proteins within the following combinations, as carrier proteins or as free proteins or mixture of the two. For example, in a combination of two proteins as set forth below, both proteins can be used as carrier proteins, or both proteins can be present as free proteins, or both can be present as carrier proteins and as free proteins, or one can be present as a carrier protein and a free protein while the other is present only as a carrier protein or only as a free protein, or one may be present as a carrier protein and the other as a free protein. Where there is a combination of three proteins, similar possibilities exist. Preferred combinations include, but are not limited to, PhtD + NR1xR2, PhtD + NR1 xR2-Sp91 Chimeric Cterm or fusion proteins, PhtD + PIy, PhtD + Sp128, PhtD + PsaA, PhtD + PspA, PhtA + NR1xR2, PhtA + NR1 xR2-Sp91 Chimeric Cterm 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 CbpA or PspC. Preferably it is CbpA. Other combinations include 3 combinations of proteins such as PhtD + NR1xR2 + PIy, and PhtA + NR1xR2 + PhtD. In one embodiment, the vaccine composition comprises detoxified pneumolysin and PhtD or PhtDE as carrier proteins. In another embodiment, the vaccine composition comprises detoxified pneumolysin and PhtD or PhtDE as free proteins. The present invention further provides a vaccine containing the immunogenetic compositions of the invention and a pharmaceutically acceptable excipient. The vaccines of the present invention may have adjuvants, particularly when they are intended for use in a population of the elderly but also for use in child populations. Suitable adjuvants include an aluminum salt such as aluminum hydroxyl gel or aluminum phosphate or alum, but may also be a calcium, magnesium, iron or zinc salt, or may be an insoluble suspension of acylated tyrosine, or acylated sugars , cationic or aniconically derived saccharides, or polyphosphazenes. It is preferred that the adjuvant be selected to be a preferential inducer of a TH1 type of response. Such levels of Th1-type cytokines tend to favor the induction of cell-mediated immune responses to a given antigen, while Th2-type cytokine levels tend to favor the induction of humoral immune responses to the antigen. The distinction between Th1 and Th2 immune responses is not absolute. In fact, an individual will support an immune response that is described as predominant Th1 or predominant Th2. However, it is often convenient to consider the cytokine families in terms of those described in murine CD4 + ve T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman, R.L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. (Annual Review of Immunology, 7, p145-173). Traditionally, Th1 type responses are associated with the production of INF-? Cytokines. and IL-2 by T lymphocytes. Other cytokines frequently associated directly with the induction of Th1 type immune responses are not produced by T cells, such as IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10. Suitable adjuvant systems that predominantly promote a Th1 response include: monophosphoryl lipid A or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (for preparation see GB 2220211 A ); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt (for example aluminum phosphate or aluminum hydroxyl) or an oil / water emulsion. In such combinations, the antigen and 3D-MPL are contained in the same particle structures, allowing a more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL can further improve the immunogenicity of an antigen absorbed by alum [Thoelen et al. Vaccine (1998) 16: 708-14; EP 689454-B1]. An improved system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where QS21 is stopped with cholesterol as described in WO 96/33739. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil / water emulsion is described in WO 95/17210. In one embodiment the immunogenetic composition further comprises a saponin, which may be QS21. The formulation may also comprise an oil / water emulsion and a tocopherol (WO 95/17210). Unmethylated CpG containing oligonucleotides (WO 96/02555) and other immunomodulatory oligonucleotides (WO0226757 and WO03507822) are also preferential inducers of a Th1 response and are suitable for use in the present invention. Particular adjuvants are those selected from the group of metal salts, oil / water emulsions, Toll-like receptor agonist, (particularly the Toll-like receptor 2 agonist, Toll-like receptor 3 agonist, similar receptor 4 agonist a Toll, Toll-like receptor 7 agonist, Toll-like receptor 8 agonist and Toll-like receptor 9 agonist), saponins or combinations thereof. An adjuvant that can be used with the vaccine compositions of the invention are outer membrane vesicle preparations or ampoules of gram-negative bacterial strains such as those taught by WO02 / 09746 - particularly ampules of N. meningitidis. The adjuvant properties of the ampoules can be improved by keeping LOS (lipooligosacaride) on its surface (for example through extraction with low concentrations of detergent [eg 0-0.1% deoxycholate]). The LOS can be detoxified Through the mutations msbB (-) or htrB (-) discussed in WO02 / 09746 Adjuvant properties can also be improved by preserving PorB (and optionally eliminating PorA) from meningococcal blisters. Adjuvant properties can also be improved by truncating the base saccharide structure of LOS in meningococcal bullae-for example via the IgtB (-) mutation discussed in WO2004 / 014417 Alternatively, the aforementioned LOS (eg isolated from a strain msbB (-) and / or IgtB (-)) can be purified and to be used as adjuvant in the compositions of the invention An additional adjuvant that can be used with the compositions of the invention can be selected from the group saponin, lipid A or derivative thereof, immunostimulatory oligonucleotide, alkyl phosphate glucosaminide, oil / water emulsion or combinations thereof Another preferred adjuvant is a metal salt in combination with another adjuvant It is preferred that the adjuvant be a Toll-like receptor agonist, particularly a 2, 3 receptor agonist, 4, 7, 8 or 9 similar to Toll, or a saponin, particularly Qs21 It is more preferred that the adjuvant system comprises two or more adjuvants of the above list Particularly the combinations preferably contain a saponin adjuvant (particularly Qs21) and / or a Toll-like receptor 9 agonist such as a CpG containing the immunostimulatory oligonucleotide. Other preferred combinations comprise a saponin (particularly QS21) and a Toll-like receptor 4 agonist such as monophosphoryl lipid A or its 3-deacylated derivative, 3D-MPL, or a saponin (particularly QS21) and a Toll-like receptor 4 ligand such as an alkyl phosphate glucosaminide. Particularly preferred adjuvants are combinations of 3D-MPL and QS21 (EP 0 671 948 B1), oil / water emulsions comprising 3D-MPL and QS21 (WO 95/17210, WO 98/56414), or 3D-MPL formulated with other carriers (EP 0 689 454 B1). Other preferred adjuvant systems comprise a combination of 3 D MPL, QS21 and a CpG oligonucleotide as described in US6558670, US6544518. In one embodiment the adjuvant is (or comprises) a ligand of Toll-like receptor 4 (TRL), preferably an agonist such as a lipid A derivative, particularly monophosphoryl lipid A or more particularly deacetylated monophosphoryl lipid A (3). D-MPL). 3 D-MPL is available from GlaxoSmithKine Biologicals North America and primarily promotes CD4 + T cell responses with an IFN-g (Th1) phenotype. It can be produced according to the methods described in GB 2 220211 A. Chemically it is a mixture of a 3-deacetylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Preferably 3 D MPL of small particle is used in the compositions of the present invention. 3 D-MPL small particle has a size Such a particle can be filtered sterile through a 0.22 μm filter. Such preparations are described in International Patent Application No. WO 94/21292. Synthetic derivatives of lipid A are known and thought to be the TLR 4 agonists including, but not limited to: OM 174 (2-deoxy-6-o- [2-deoxy-2 - [(R) - 3-dodecanoyloxytetra-decanoylamino] -4-o-phosphono-pD-glucopyranosyl] -2 - [(R) -S-hydroxy-tetraalkylamino] -D-glucopyranosyldihydrogen phosphate), (WO 95/14026) OM 294 DP (3S , 9R) -3 - [(R) -dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 (R) - [(R) -3-hydroxytetradecanoylamino] decan-1, 1 Od io 1, 1, 10-b ? s (dihydrogen phosphate) (WO 99/64301 and WO 00/0462) OM 197 MP-Ac DP (3S, 9R) -3 - [(R) -dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 - [( R) -3-hydroxytetradecanoylamino] decan-1,10-diol, 1-dihydrogenphosphate-10- (6-aminohexanoate) (WO 01/46127) Other ligands of TLR4 which can be used are alkyl phosphate glucosaminide such as those described in WO9850399 or US6303347 (the processes for the preparation of AGPs are also described), or pharmaceutically acceptable salts of AGPs as described in US6764840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. Both are probably 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). The purified fragments of Quil A have been isolated with HPLC which retains the adjuvant activity without 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 the CD8 + T cytotoxic cells (CTLs), Th1 cells and a predominant response of the IgG2a antibody and is a preferred saponin in the context of the present invention. It has been described that the particular formulations of QS21 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 be in the form of ISCOM matrices (EP 0 109 942 B1), liposomes or structures related colloids such as worm-shaped or ring-shaped multimeric complexes or lipid structures / with layers and lamellae when formulated with cholesterol and lipid, or in the form of an oil / water emulsion (for example as in WO 95/17210). The saponins can be preferably associated with a metal salt, such as aluminum hydroxide or aluminum phosphate (WO 98/15287). Preferably, the saponin is presented in the form of liposome, ISCOM or oil / water emulsion. An improved system involves the combination of a monophosphoryl lipid A (or detoxified lipid A) and a saponin derivative, particularly the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where QS21 is stop with cholesterol as described in WO 96/33739. An adjuvant formulation particularly involving tocopherol with or without QS21 and / or 3D-MPL in an oil / water emulsion is described in WO 95/17210. In one embodiment the immunogenetic composition further comprises a saponin, which may be QS21. Immunostimulatory oligonucleotides or any other Toll-like receptor 9 agonist can also be used. Preferred oligonucleotides for use in adjuvants or vaccines of the present invention are oligonucleotides containing CpG, preferably containing two or more CpG motifs of dinucleotide separated by at least three, preferably at least six or more nucleotides. A CpG motif is a cytosine nucleotide after 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 the phosphodiester 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 phosphorodithioate or phosphorothioate oligonucleotides are described in US5,666,153, US5,278,302 and WO95 / 26204. Examples of preferred oligonucleotides have the following sequences. The sequences preferably contain phosphorothioate-modified internucleotide linkages. OLIGO 1 (SEQ ID NO: 1): TCC ATG ACG TTC CTG ACG TT (CpG 1826) OLIGO 2 (SEQ ID NO: 2): TCT CCC AGC GTG CGC CAT (CpG 1758) OLIGO 3 (SEQ ID NO: 3) : ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG OLIGO 4 (SEQ ID NO: 4): TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) OLIGO 5 (SEQ ID NO: 5): TCC ATG ACG TTC CTG ATG CT (CpG 1668) OLIGO 6 (SEQ ID NO: 6): TCG ACG TTT TCG GCG CGC GCC G (CpG 5456) Alternative CpG oligonucleotides can comprise the preferred sequences above where they have 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, such oligonucleotides can be synthesized using an automated synthesizer. The adjuvant may be an oil / water emulsion or may comprise an oil / water emulsion in combination with other adjuvants. The oil phase of the emulsion system preferably comprises a metabolisable 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 oil, fish oil, animal or synthetic oil, which is not toxic to the recipient and is capable of being 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 lower amounts in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in this invention. He squalene is a metabolizable oil by virtue of the fact that it is an intermediate in cholesterol biosynthesis (Merck index, 1 Oth Edition, entry no.8619). Touches (for example vitamin E) are also frequently used in oil emulsion adjuvants (EP 0 382 271 B1, US5667784, WO 95/17210). The knobs used in the oil emulsions (preferably oil / water emulsions) of the invention may be formulated as described in EP 0 382 271 B1, where the knobs may be dispersions of tocol droplets, optionally comprising an emulsifier, preferably less than 1 micron in diameter. Alternatively, the knobs may be used in combination with another oil, to form the oil 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. Oil / water emulsion adjuvants by themselves have been suggested to be useful as adjuvant compositions (EP 0 399 843B), also combinations of oil / water emulsions and other active agents have been described as adjuvants for vaccines (WO 95/17210, WO 98/56414, WO 99/12565, WO 99/11241). Other oil emulsion adjuvants have been described, for example water in the oil emulsions (US 5,422,109; EP 0 480 982 B2) and water in the emulsions of oil / water (US 5,424,067, EP 0 480 981 B). All form preferred oil emulsion systems (particularly when incorporating touches) to form the adjuvants and compositions of the present invention. More preferably the oil emulsion (for example oil / water emulsions) additionally comprises an emulsifier such as TWEEN 80 and / or a sterol such as cholesterol. A preferred oil emulsion (preferably oil / water emulsion) comprises a non-toxic metabolizable oil, such as squalane, squalene or tocopherol such as tocopherol alfa (and preferably squalene and alpha tocopherol) and optionally an emulsifier (or surfactant) such as Tween. 80. A sterol (preferably cholesterol) may also be included. The method for producing oil / 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 PBS / TWEEN80 ™ solution, followed by homogenization using a homogenizer, it would be clear to one skilled in the art that a method comprising Twice the mixing through a syringe needle would be convenient to homogenize small volumes of liquid. Also, the emulsification process in the microfluidizer (M110S Microfluidics machine, maximum of 50 steps, during a period of 2 minutes at the maximum pressure input of 6 bar (outlet pressure of approximately 850 bar)), could be adapted by the person skilled in the art to produce smaller or larger volumes of emulsion. The adaptation could be achieved by routine experimentation comprising the measurement of the resulting emulsion until a preparation was achieved with oil droplets of the required diameter. In an oil / water emulsion, the oil and the emulsifier must be in an aqueous carrier. The aqueous carrier can be, for example, saline solution buffered with phosphate. The size of the oil droplets found in the stable oil / water emulsion is preferably less than 1 micron, may be in the range of substantially 30-600 nm, preferably substantially about 30-500 nm in diameter, and more preferably substantially 150-500 nm in diameter, and particularly about 150 nm in diameter as measured by photon correlation spectroscopy. In this regard, 80% of the oil drops per number must be within the preferred ranges, preferably more than 90% and more preferably more than 95% of the oil drops per number are within the defined ranges of size. The amounts of components present in the oil emulsions of the present invention are conventionally in the range of 0.5-20% or 2 to 10% oil (total dose volume), such as squalene; and when present, from 2 to 10% tocopherol alpha, and from 0.3 to 3% surfactant, such as polyoxyethylene sorbitan monooleate. Preferably the ratio of oil (preferably squalene): tocol (preferably α-tocopherol) is equal to or less than 1 since this provides a more stable emulsion. An emulsifier, such as TweendO or Span 85 may also be present at a level of about 1%. In some cases it may be advantageous if the vaccines of the present invention additionally contain a stabilizer. Examples of the preferred emulsion systems are described in WO 95/17210, WO 99/1 1241 and WO 99/12565 which describe the emulsion adjuvants based on squalene, α-tocopherol, and TWEEN 80, optionally formulated with immunostimulants QS21 and / or 3D-MPL. Thus in a particularly preferred embodiment of the present invention, the adjuvant of the invention may further comprise other Immunostimulants, such as LPS or derivatives thereof, and / or saponins. Examples of other immunostimulants are described herein and in "Vaccine Design - The Subunit and Adjuvant Approach" 1995, Pharmaceutical Biotechnology, Volume 6, Eds Powell, M. F., and Newman, M. J., Plenum Press, New York and London, ISBN 0-306-44867-X. In a preferred aspect, the adjuvant and immunogenetic compositions according to the invention comprise na saponin (preferably QS21) and / or an LPS derivative (preferably 3D-MPL) in an oil emulsion described above, optionally with a sterol (preferably cholesterol). Additionally, the oil emulsion (preferably oil / water emulsion) may contain Span 85 and / or lecithin and / or tricaprylin. Adjuvants comprising an oil / water emulsion, a sterol and a saponin are described in WO 99/12565. Commonly for human administration saponin (preferably QS21) and / or LPS derivatives (preferably 3D-MPL) will be present at a human dose of immunogenic composition in the range of 1 μg-200 μg, such as -100 μg, preferably 10 μg-50 μg per dose. Commonly the oil emulsion (preferably emulsion-oil / water) will comprise from 2 to 10% metabolizable oil. Preferably it will comprise from 2 to 10% squalene, from 2 to 10% tocopherol alfa and from 0.3 to 3% emulsifier (preferably 0.4-2%) (preferably tween 80 [polyoxyethylene sorbitan monooleate]). Where squalene and alpha tocopherol are present, preferably the squalene: tocopherol alpha ratio is equal to or less than 1 since it provides a more stable emulsion. Span 85 (sorbitan trioleate) may also be present at a level of 0.5 to 1% in the emulsions used in the invention. In some cases it may be advantageous that the immunogenetic compositions and vaccines of the present invention additionally contain a stabilizer, for example other emulsifiers / surfactants, including caprylic acid (Merck Index 10th Edition, entry No. 1739), whose tricaprylin is particularly preferred. Where squalene and a saponin (preferably QS21) are included, it is also advantageous to include a sterol (preferably cholesterol) to the formulation while permitting a reduction in the total level of oil in the emulsion. This leads to a reduced manufacturing cost, to the improvement of the total adaptation of the vaccination, and also to the qualitative and quantitative improvements of the resulting immune responses, such as the production of IFN-α. improved Accordingly, the adjuvant system of the present invention commonly comprises a ratio of metabolizable oil: saponin (w / w) in the range of 200: 1 to 300: 1, also the present invention can be used in a "low oil" form ", whose preferred range is 1: 1 to 200: 1, preferably 20: 1 to 100: 1, and most preferably substantially 48: 1, this vaccine retains the beneficial adjuvant properties of all components, with a reactogenicity profile very reduced. Accordingly, particularly preferred embodiments have a squalene ratio: QS21 (w / w) in the range of 1: 1 to 250: 1, also a preferred range is 20: 1 to 200: 1, preferably 20: 1 to 100 : 1, and more preferably substantially 48: 1. Preferably a sterol (more preferably cholesterol) is also included at a ratio of saponin: sterol as described herein.

The emulsion systems of the present invention preferably have a small drop size of oil in the sub-micron range. More preferably the oil drop sizes will be in the range of from 120 to 750 nm, and more preferably from 120-600 nm in diameter. A particularly potent adjuvant formulation (for the latter combination with AIPO4 in the immunogenetic compositions of the invention) involves a saponin (preferably QS21), an LPS derivative (preferably 3D-MPL) and an oil emulsion (preferably squalene and alpha tocopherol in an oil / water emulsion) as described in WO 95/17210 or WO 99/12565 (particularly the adjuvant formulation 11 in Example 2, Table 1). Examples of a TLR2 agonist include peptidoglycan or lipoprotein. Imidazoquinolines, such as Imiquimod and Resiquimod, are known TLR7 agonists. The single-stranded RNA is also a known TLR agonist (TLR8 in humans and TLR7 in mice), while dobel filament RNA and poly IC (polyinosinic-polycytidyl acid - a synthetic commercial mimic of viral RNA) are examples of agonists of TLR 3. 3D-MPL is an example of a TLR4 agonist while CPG is an example of a TLR9 agonist. The immunogenetic composition may comprise an antigen and an immunostimulant absorbed in a metal salt. Aluminum-based vaccine formulations where the antigen and 3-de-O-acylated monophosphoryl lipid A (3D-MPL) immunostimulant, are absorbed in the same particle, are described in EP 0 576 478 B1, EP 0 689454 B1, and EP 0 633 784 B1. In these cases then the antigen is first absorbed into the aluminum salt followed by the adsorption of immunostimulatory 3D-MPL on the same aluminum salt particles. Such processes first involve the suspension of 3D-MPL by the sonic treatment in a water bath until the particles reach a size between 80 and 500 nm. The antigen is commonly absorbed in the aluminum salt for one hour at room temperature under agitation. The 3D-MPL suspension is then added to the absorbed 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 metal particles, as described in EP 1126876. The improved process comprises the adsorption of immunostimulant, in a metal salt particle, followed by the adsorption of the antigen in another metal particle of salt, followed by the mixing of discrete metallic particles to form a vaccine. The adjuvant for use in the present invention can be an adjuvant composition comprising an immunostimulant, absorbed in a metal salt particle, characterized in that the metal salt particle is substantially free of another antigen. In addition, vaccines are provided by the present invention and characterized in that the immunostimulant is absorbed in the metal salt particles that are substantially free of another antigen, and wherein the metal salt particles that are absorbed in the antigen are substantially free of another immunostimulant.

Accordingly, the present invention provides an adjuvant formulation comprising an immunostimulant that has been absorbed into a metal salt particle, wherein the composition is substantially free of another antigen. On the other hand, this adjuvant formulation can be an intermediate which, if such an adjuvant is used, is required for the manufacture of a vaccine. Accordingly, a process for the manufacture of a vaccine comprising mixing an adjuvant composition that is one or more immunostimulants absorbed in a metal particle with an antigen is provided.

Preferably, the antigen has been pre-absorbed in a metal salt. The metal salt may be identical or similar to the metal salt that is absorbed in the immunostimulant. The metal salt is preferably an aluminum salt, for example aluminum phosphate or aluminum hydroxyl. The present invention further provides a vaccine composition comprising an immunostimulant absorbed in a first particle of a metal salt, and an antigen absorbed in a metal salt, wherein the first and second metal salt particles are separate particles.

The LPS or LOS derivatives or the mutations or lipid A derivatives described herein are designed to be less toxic (e.g. 3D-MPL) than the native lipopolysaccharides and are interchangeable equivalents with respect to any use of these portions described herein. They can be ligands of TLR4 as described above. Other derivatives are described in WO020786737, WO9850399, WO0134617, WO0212258, WO03065806. In one embodiment the adjuvant used for the i or compositions of the invention comprises a liposome carrier (made by known phospholipid techniques (such as phospholipid dioleoyl choline [DOPC]) and a sterol [such as cholesterol]). Such liposome carriers may optionally carry lipid A derivatives [such as 3D-MPL - see above] and / or saponins (such as QS21 - see above). In one embodiment the adjuvant comprises (per 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) phospholipids (for example DOPC), 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), 5-60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10 , 20, 30, 40 or 50 μg) derived from lipid A (for example 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). 25 This adjuvant is particularly suitable for vaccine formulations for the elderly. In one embodiment, the vaccine composition comprising this adjuvant encompasses the saccharide conjugates derived from at least all of the following serotypes 4, 6B, 9V, 14, 18C, 19F1 23F, 1, 5, 7F (and may also comprise one or more of serotypes 3, 6A, 19A, and 22F), wherein 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 that induced by the Prevnar® vaccine in human receptors. In one embodiment, the adjuvant used for the compositions of the invention comprises an oil / water emulsion made from a metabolizable oil (such as squalene), an emulsifier (such as tween 80) and optionally a tocol (such as alpha tocopherol). In one embodiment the adjuvant comprises (per 0.5 ml dose) 0.5-15, 1-13, 2-1 1, 4-8, or 5-6 mg (eg 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 (eg 0.9-1-1, 2-3 or 4-5 mg) 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) tocol (such as alpha tocopherol). This adjuvant can optionally further comprise 5-60, 10-50, or 20-30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) derived from lipid A (e.g. 3D- MPL). These adjuvants are particularly suitable for vaccine formulations for infants or the elderly. In a embodiment the vaccine composition comprising this adjuvant comprises saccharide conjugates derived from at least all of the following serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more of serotypes 3, 6A, 19A, and 22F), where the antibody titer of GMC induced against one or more (or all) vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferior to that induced by the Prevnar® vaccine in human receptors. This adjuvant can optionally contain 0.025-2.5, 0. 05-1.5, 0.075-0.75, 0.1-0.3, or 0.125-0.25 (eg 0.2-0.3, 0.1-0.15, 0.25-OR 0.125 mg) sterol (eg cholesterol), 5-60, 10-50, or 20 -30 μg (for example 5-15, 40-50, 10, 20, 30, 40 or 50 μg) derived from lipid A (for example 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 comprising this adjuvant comprises the saccharide conjugates derived from at least all of the following serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more of serotypes 3, 6A, 19A, and 22F), wherein 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 human receptors. In one embodiment the adjuvant used for the compositions of the invention comprises aluminum phosphate and a lipid A derivative (such as 3D-MPL). This adjuvant may comprise (per dose of 0.5 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) derived from lipid A (for example 3D-MPL). This adjuvant is particularly suitable for vaccine formulations for the elderly or infants. In one embodiment the vaccine composition comprising this adjuvant comprises the saccharide conjugates derived from at least all of the following serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more of serotypes 3, 6A, 19A, and 22F), where the antibody titer of GMC induced against one or more (or all) vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than that induced by the Prevnar® vaccine in human receptors. The vaccine preparations containing the immunogenetic compositions of the present invention can be used to protect or treat a mammal susceptible to infection, by administering the vaccine via a systemic or mucosal route. These administrations may include injection via intramuscular routes, intraperitoneal, intradermal or subcutaneous, or via administration through the mucosa to the oral / alimentary, respiratory, genitourinary tracts. Intranasal administration of vaccines for the treatment of pneumonia or otitis media is preferred (since the nasopharyngeal vehicle of pneumococci can be prevented more effectively, thus attenuating the infection in its first 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 pneumococcal saccharide conjugates could be administered separately, at the same time or 1-2 weeks after the administration of any bacterial protein component of the vaccine for optimal coordination of the immune responses with respect to each other). For co-administration, the optional Th1 adjuvant can be present in any or all of the different administrations. In addition to a single administration route, 2 different administration routes can be used. For example, saccharides or saccharide conjugates can be administered IM (or ID) and bacterial proteins can be administered IN (or ID). In addition, the vaccines of the invention can be administered IM for the first doses and IN for the booster doses. The content of protein antigens in the vaccine will commonly be in the range of 1-100 μg, preferably 5-50 μg, most commonly in the range of 5-25 μg. After an initial vaccination, subjects may receive one or several booster immunizations spaced appropriately. The vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell M. F. &Newman M.J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, US Patent No. 4,235,877. The vaccines of the present invention can be stored in solution or lyophilized. The solution is preferably lyophilized in the presence of a sugar such as sucrose or lactose. Furthermore, it is even more preferable that they be lyophilized and reconstituted extemporaneously before use. Lyophilization can result in a more stable composition (vaccine) and 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 there is provided a vaccine kit, comprising a bottle containing an immunogenetic composition of the invention, optionally in lyophilized form, and further comprising a bottle containing an adjuvant as described herein. It is provided that in this aspect of the invention, the adjuvant is used to reconstitute the lyophilized immunogenic composition. Although the vaccines of the present invention can be administering by any route, administration of the described skin (ID) vaccines forms one embodiment of the present invention Human skin comprises an outer "corneous" cuticle, called stratum corneum, which covers the epidermis. Below this epidermis is a layer called the dermis, which in turn covers the subcutaneous tissue. Researchers have shown that injection of a vaccine into the skin, and particularly into the dermis, stimulates an immune response, which can also be associated with a number of additional benefits. Intradermal with the vaccines described in this form a preferred feature of the present invention The conventional technique of intradermal injection, the "Mantoux procedure", comprises the steps of cleaning the skin, and then stretching it with one hand, and with the bevel of a narrow gauge needle (caliber 26-31) that is opposite upwardly of the needle, inserted at an angle between 10-15 ° Since the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced while providing a slight pressure to raise it under the skin. The liquid is then injected in such a way that it forms very slightly through the skin. same a blister or bulge on the surface of the skin, followed by the slow removal of the needle More recently, devices that are specifically designed to deliver liquid agents to or through the skin, for example the devices described in WO, have been described. 99/34850 and EP 1092444, also the jet injection devices described for example in WO 01/13977; US 5,480,381, US 5,599,302, US 5,334,144, US 5,993,412, US 5,649,912, US 5,569,189, US 5,704,911, US 5,383,851, US 5,893,397, US 5,466,220, US 5,339,163, US 5,312,335, US 5,503,627, US ,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 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 applied to the surface of the skin (transdermal or transcutaneous supply 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 is at a low liquid volume, particularly a volume 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 conventional doses as found in intramuscular vaccines (see above). However, it is a characteristic of skin or intradermal vaccines that the formulations may be "low dose". Therefore protein antigens in "low dose" vaccines are preferably present in portions as small as 0.1 to 10 μg, preferably 0.1 to 5 μg per dose; and saccharide antigens (preferably conjugates) 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. Generally 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 cornea and the epidermis on the surface and the subcutaneous layer below. Depending on the mode of delivery, the vaccine may ultimately be located only or mainly within the dermis, or may ultimately be distributed within the epidermis and dermis. The present invention also provides an improved vaccine for the prevention or amelioration of the means of Otitis caused by Hemophilus influenzae by the addition of proteins of Hemophilus influenzae, for example protein D in free or conjugated form. In addition, the present invention additionally provides an improved vaccine for the prevention or amelioration of pneumococcal infection in infants (eg, Otitis media), based on the addition of one or two pneumococcal proteins as free or conjugated protein to the conjugate compositions of S. pneumoniae of the invention. The pneumococcal free proteins may be the same as or different from any protein of S. pneumoniae used as a carrier protein. One or more Moraxella catarrhalis protein antigens can also be included in the combination vaccine in a free or conjugated form. Thus, the present invention is an improved method for producing an immune (protective) immune response against Otitis media in children. In another embodiment, the present invention is an improved method for producing an immune (protective) immune 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 the invention [a pediatric vaccine]. Other embodiments of the present invention include the provision of antigenic conjugate compositions of S. pneumoniae of the invention for use in medicine and for the use of the conjugates of S. pneumoniae of the invention in the manufacture of a medicament for the prevention ( or treatment) of pneumococcal disease. In yet another embodiment, the present invention is an improved method for producing an immune (protective) immune response in the elderly population (in the context of the present invention, consider an elderly patient if he is 50 years of age or older, commonly more than 55 years and more generally more than 60 years old) administering a safe and effective amount of the vaccine of the invention, in combination with one or two S proteins. pneumoniae preferably present as free or conjugated protein, whose S. pneumoniae free proteins may be the same as or different from any S. pneumoniae protein used as a carrier protein. A further aspect of the invention is a method for immunizing a human host against the disease caused by S. pneumoniae and optionally the infection of Hemophilus influenzae, which comprises administering to the host a dose, immunoprotective of the composition or immunogenic vaccine or kit of the invention. . A further aspect of the invention is an immunogenetic composition of the invention for use in the treatment or prevention of the disease caused by S. pneumoniae and optionally the infection of Hemophilus influenzae. A further aspect of the invention is the use of the immunogenetic 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 the infection of Hemophilus influenzae. The terms "comprising", "comprises" and "comprise" in the present are intended by the inventors as optionally replaceable with the terms "consisting of, "consist of" and "consist of", respectively, in each case. The modalities in the present that refer to the "Vaccine compositions" of the invention are also applicable to the modalities relating to the "immunogenetic compositions" of the invention, and vice versa. All references or Patent Requests cited within this Patent Specification are incorporated by reference herein. In order to better understand this invention, the following examples are provided. These examples are for illustration purposes only, and in no way should they be construed as limiting the scope of the invention. Examples Example 1: Expression of protein D Protein D of Hemophilus influenzae Genetic construction for the expression of protein D Starting materials DNA coding for protein D Protein D is highly conserved among H. influenzae of all serotypes and unclassifiable strains. The vector pHIC348 containing the DNA sequence encoding the entire protein D gene has been obtained from Dr. A Forsgren, Department of Medical Microbiology, University of Lund, Malmo General Hospital, Malmo, Sweden. The DNA sequence of protein D has been published by Janson et al. (1991) Infect Immun 59: 119-125. Expression vector pMG1 The pMG1 vector of the expression is a derivative of pBR322 (Gross et al., 1985) where the control elements derived from the bacteriophage were introduced? for the transcription and translation of foreign inserted genes (Shatzman et al., 1983). In addition, the ampicillin resistance gene was exchanged for the kanamycin resistance gene. E. Coli strain AR58 The E. coli strain AR58 was generated by the transduction of N99 with a bacteriophage P1 portion previously developed in an SA500 derivative (galE :: TN10, lambdaKil cl857? H1). N99 and SA500 are strains of E. coli K12 derived from Dr. Martin Rosenberg's laboratory at the National Institute of Health.

Expression vector pMG 1 For the production of protein D, the DNA encoding the protein has been cloned into the expression vector pMG 1. This plasmid uses lambdafago DNA signals to drive the transcription and translation of inserted foreign genes. The vector contains the PL promoter, OL operator and two sites of use (NutL and NutR) to decrease the effects of polarity transcription when the N protein is provided (Gross et al., 1985). The vectors containing the PL promoter are introduced into a lysogenic host of E. coli for stabilize the plasmid DNA. The strains of the lysogenic host contain the lambdafago DNA defective to the replication integrated into the genome (Shatzman et al., 1983). The chromosomal lambdafago DNA directs the synthesis of the repressor protein cl that binds to the OL repressor of the vector and prevents the binding of the RNA polymerase to the PL promoter and thereby the transcription of the inserted gene. The cl gene of expression strain AR58 contains a thermosensitive mutant so that PL-directed transcription can be regulated by the change in temperature, i.e. an increase in culture temperature inactivates the repressor and protein synthesis is initiated foreign This expression system allows 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 of E. coli AR58 used for the production of the protein carrier D is a derivative of the NIH strain E. coli K12 standard N99 (F "its" galK2, lacZ "thr"). Contains a defective lysogenic lambdafago (galE :: TN10, lambdaKil "cl857? H1). Kil" phenotype prevents the inhibition of macromolecular synthesis of the host. The cl857 mutation confers a thermosensitive lesion to the cl repressor. The suppression of? H1 eliminates the right operon of the lambdafago and the bio location, uvr3, and chIA of the hosts. Strain AR58 was generated by the transduction of N99 with a portion of phage P1 developed previously in an SA500 derivative (galE :: TN10, lambdaKM "cl857? H1). The introduction of the defective lysogen in N99 was selected with tetracycline by virtue of the presence of a TN 10 transposon coding for the tetracycline resistance in the gene adjacent galE Construction of vector pMGMDPPrD The 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 of vector pHIC348 ( Janson et al 1991 Infect, Immun 59: 119-125) with the PCR primers containing the Ncol and Xbal restriction sites at 5 'and 3' ends, respectively. The Ncol / Xbal fragment was then introduced into pMGNSI between Ncol and Xbal, thus creating a fusion protein containing 81 N-terminal amino acids of the NS1 protein followed by the PD protein. This vector was labeled pMGNSIPrD. Based on the construct described above, the final construct was generated for the expression of protein D. A fragment of BamHI / BamHI was removed from pMGNSIPrD. This DNA hydrolysis removes the coding region of NS1, except for the first three N-terminal residues. During the religation of the vector, a gene encoding a fusion protein with the following N-terminal amino acid sequence has been generated: MDP SSHSSNMANT NS1 protein D Protein D does not contain an N-terminal leader peptide or cysteine whose lipid chains bind normally. The protein is therefore not excreted in the periplasm or lipid and remains in the cytoplasm in a soluble form. The final construct pMG-MDPPrD was introduced into the AR58 strain of the host by heat shock at 37 ° C. The plasmid containing the bacteria was selected in the presence of kanamycin. The presence of the DNA insert encoding protein D was demonstrated by the digestion of DNA isolated from the plasmid with the selected endonucleases. The recombinant strain of E. coli is referred to as ECD4. The expression of protein D is under the control of the PL promoter / O lambda operator. The AR58 strain of the host contains a thermosensitive cl gene in the genome that blocks the expression of lambda P at low temperature by binding to OL. Once the temperature rises, it is released from OL and protein D is expressed. Small scale preparation At the end of the fermentation, the cells are concentrated and frozen. The extraction of the harvested cells and the purification of protein D were carried out as follows. The frozen cell culture granule is thawed and is suspended again in a Cell alteration solution (citrate buffer, pH 6.0) at a final OD650 = 60. The suspension is passed twice through a high pressure homogenizer at P = 1000 bar. The cell culture homogenate is purified by centrifugation and the cell waste is removed by filtration. In the first purification step the used filtrate is applied to a cation exchange chromatography column (SP sepharose fast flow). PD binds to the gel matrix by ionic interaction and is eluted by an increase in the ionic strength of the elution buffer. In a second purification step, the impurities are conserved in an ammonium exchange matrix (fast flow sepharose Q). PD does not bind to the gel and can be collected in the direct flow. In both steps of column chromatography, fraction collection is monitored by OD. The direct flow of the anion exchange column chromatography containing the purified protein D is concentrated by ultrafiltration. Protein D containing the ultrafiltration retentate is finally passed through 0.2 μm membranes. Large-scale preparation The extraction of harvested cells and the purification of protein D were carried out as follows. The harvested broth is cooled and passed directly twice through a high pressure homogenizer at a pressure of approximately 800 bar. In the first purification step, the cell culture homogenate is diluted and applied to a cation exchange chromatography column (large SP Sepharose granules). PD binds to the gel matrix by ionic interaction and is eluted by an increase in the ionic strength of the elution buffer and filtered. In a second purification step, the impurities are conserved in an anion exchange matrix (rapid flow of Sepharose Q). PD does not bind to the gel and can be collected in the direct flow. In both steps of column chromatography, fraction collection is monitored by OD. The direct flow of the anion exchange column chromatography containing the purified protein D is concentrated and diafiltered by ultrafiltration.

Protein D containing the ultrafiltration retentate is finally passed through a 0.2 μm membrane. Example 1b: Expression of PhtD The PhtD protein is a member of the pneumococcal histidine protein family (Pht) characterized by the presence of histidine triads (motif HXXHXH). PhtD is an 83S aa molecule and carries 5 triads of histidine (see Medlmmune WO00 / 37105 SEQ ID NO: 4 for the amino acid sequence and SEQ ID NO: 5 for the DNA sequence). PhtD also contains a proline-rich region in the center (amino acid position) 348-380). PhtD has a 20 aa-N-terminal signal sequence with a LXXC motif. Genetic construct The genetic sequence of the mature PhtD protein of Medlmmune (from aa 21 to a838) was transferred recombinantly to E. coli using the internal vector pTCMP14 carrying the p ?. The E. coli strain of the host is AR58, which carries the clone-sensitive repressor, allowing the heat induction of the promoter. The polymerase chain reaction was observed to amplify the phtD gene of a Medlmmune plasmid (carrying the phtD gene of Streptococcus pneumoniae Norway 4 strain (serotype 4) - SEQ ID NO: 5 as described in WO 00/37105 ). The primers specific for the phtD gene alone were used to amplify the phtD gene in two fragments. The primers carry Ndel and Kpnl or the restriction sites Kpnl and Xbal. These primers do not hybridize with any nucleotide of the vector but only with the specific sequences of the phtD gene. An artificial ATG start codon was inserted using the first primer carrying the Ndel restriction site. The PCR generated products were then inserted into the cloning vector pGEM-T (Promega), and the DNA sequence was confirmed. The sub-cloning of the fragments in the TCMP expression vector 14 was then observed using standard techniques and the vector was transformed to AR58 E. coli.

Purification of PhtD The purification of PhtD is achieved as follows: • Growth of E. coli cells in the presence of kanamycin: growth of 30 hours at 30 ° C followed by induction for 18 hours at 39.5 ° C • Separation of cells from E. coli of the whole culture at OD ± 115 in the presence of 5 mM EDTA and 2 mM PMSF as protease inhibitors: Rannie, 2 steps, 1000 bar. • Antigen capture and elimination of cellular waste in Streamiine Q XL chromatography expanded in bed mode 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 + 0.25% Empigen in 25 mM potassium phosphate buffer, pH 7.4. • Filtration in Sartobran 150 cartridge (0.45 + 0.2 μm) • Antigen binding in FF IMAC chromatography of Zn + + sepharose chelation at pH 7.4 in the presence of 5 mM imidazole at 4CC, the column washed with 5 mM imidazole and Empigen 1% and eluted with 50 mM imidazole, both in 25 mM potassium phosphate buffer, pH 8.0. • Weak anion exchange chromatography in positive mode in EMD DEAE Fractogel at pH 8.0 (25 mM potassium phosphate) at 4 ° C; the column is washed with 140 mM NaCl and eluted in 200 mM NaCl while the contaminants (proteins and DNA) remain absorbed in the exchanger. • Concentration and ultrafiltration with 2 mM of Na / K phosphate at pH 7.15 in 50 kDa membrane. • Sterilization filtration of the purified mass in a 0.2 μm Mili ipak-20 filter cartridge. Example 1c: Expression of pneumolysin Pneumococcal pneumolysin was prepared and detoxified as described in WO2004 / 081515 and WO2006 / 032499. Example 2: Preparation of conjugates It is well known in the art how to make purified pneumococcal polysaccharides. For the purposes of these examples the polysaccharides were made essentially as described in EP072513 or by closely related methods. Before conjugation, the polysaccharides can be sized by microfluidization as described below. The activation and coupling conditions are specific for each polysaccharide. These are given 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. The optimal polysaccharide concentration was evaluated for all serotypes. All serotypes except serotype 18C were conjugated directly to the carrier protein as detailed below. Two alternative conjugates of 22F serotypes were made; one conjugated directly, one through an ADH link.

From a stock solution of 100 mg / ml in acetonitrile or 50% / 50% acetonitrile / water solution, CDAP (ratio CDAP / PS 0.5-1.5 mg / mg PS) was added to the polysaccharide solution. 1.5 minutes later, 0.2M-0.3M NaOH was added to obtain the specific activation pH. Activation of the polysaccharide was performed at this pH for 3 minutes at 25 ° C. The purified protein (protein D, PhtD, pneumolysin or DT) (the amount depends on the initial ratio of the carrier protein / PS) was added to the activated polysaccharide and the coupling reaction was performed at specific pH for up to 2 hours (depending on serotypes) under pH regulation. To stop the unreacted cyanate ester groups, a 2M glycine solution was then added to the mixture. The pH was adjusted to buffer pH (pH 9.0). The solution was stirred for 30 minutes at 25 ° C and then overnight at 2-8 ° C with continuous slow agitation. Preparation of 18C 18C was ligated to the carrier protein via a bond-adipic acid dihydrazide (ADH). The 18C polysaccharide serotypes were 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 to reach a final concentration of 0.2M. When the separator solution was complete, the pH was adjusted to 6.2.

EDAC (1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide) was then added to reach a final concentration of 0.02M and the mixture was stirred for 1 hour under pH regulation. The condensation reaction was stopped by increasing the pH to 9.0 for at least 30 minutes at 25 ° C. TT derivative was then diafiltered (10 kDa CO membrane) to remove residual reagent from ADH and EDAC. The TTAH mas was finally sterile filtered until the coupling stage and stored at -70 ° C. Chemical coupling of TTAH to PS 18C The details of the conjugate parameters can be found in Table 1. 2 grams of microfluidized PS were diluted to the defined concentration in water and adjusted to 2M NaCl by the addition of NaCl powder. The CDAP solution (freshly prepared 100 mg / ml at 50/50 v / v acetonitrile / WFI) was added to achieve the appropriate ratio of CDAP / PS. The pH was raised to pH of activation 9.0 by the addition of 0.3M NaOH and stabilized at this pH until the addition of TTAH. After 3 minutes, TTAH derivative (20 mg / ml in 0.2 M NaCl) was added to reach a TTAH / PS ratio of 2; the pH was regulated at coupling pH 9.0. The solution was left for one hour under regulation pH. To stop the reaction, a solution of 2M glycine, was added to the PS / TTAH / CDAP mixture. The pH was adjusted to buffer pH (pH 90). The solution was stirred for 30 min at 25 ° C, and then left overnight at 2-8 ° C with continuous slow stirring. Conjugate of PS22FAH-PhtD In a second conjugation method for this saccharide (the first is the method of direct conjugation of PS22-PMD shown in Table 1), 22F was bound to the carrier protein via a bond-adipic acid dihydrazide (ADH). The 22F serotype of polysaccharide was microfluidized before conjugation. Derivation of PS 22F Activation and coupling are carried out at 25 ° C under continuous stirring in a temperature controlled water bath.

The microfluidized PS22F was diluted to obtain a final PS concentration of 6 mg / ml in 0 2M NaCl and the solution was adjusted to pH 6.05 ± 0.2 with 0.1 N HCl. The CDAP solution (100 mg / ml freshly prepared in acetonitrile / WFI, 50/50) was added to achieve the appropriate ratio of CDAP / PS (1.5 / 1 w / w). The pH was raised to activation pH 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 to reach the appropriate ratio of ADH / PS (8.9 / 1 w / w), the pH was regulated to coupling pH 9.0. The solution was left for 1 hour under regulation pH.

The PSAH derivative was concentrated and diafiltered. Coupling PhtD at 10 mg / ml in 0.2M NaCl was added to the derivative of PS22FA to reach 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 at 10 minutes (250 μl / minute) to reach 1 mg EDAC / mg PS22FAH. The resulting solution was incubated for 150 min (although 60 minutes were also used) at 25 ° C under agitation and pH regulation. The solution was neutralized by the addition of 1 M Tris-HCl pH 7.5 (1/10 final volume) and left 30 min at 25 ° C. Before elution in Sephacryl S400HR, the conjugate was purified using a 5μm Minisart filter. The resulting conjugate has a final PhtD / PS ratio of 4.1 (w / w), a PS-free content below 1% and an antigenicity (a-PS / a-PS) of 36.3% and anti-PhtD antigenicity of 7.4 %. Conjugation Purification The conjugates were purified by gel filtration using a Sephacryl S400HR gel filtration column equilibrated with 0.15M NaCl (S500HR for 18C) to remove small molecules (including DMAP) and PS and the unconjugated protein. According to the different molecular sizes of the reaction components, the PS-PD conjugates, PS-TT, PS-PhtD, PS-pneumoniae or PS-DT are eluted first, followed by free PS, then by free PD or free DT and finally DMAP and other salts (NaCl, g l icine). The fractions containing the conjugates are detected by UV280 n m- The fractions are rupaned according to their Kd, filtered sterile (0.22 μm) and stored at + 2-8 ° C. The PS / Protein ratios in the conjugate preparations were determined. Specific conditions for activating / coupling / stopping the reaction of the conjugates of PS S. pneumoniae-Protein DITTIDTlPhtD / Ply Where "μfluid" appears in the heading of the row, it indicates that the saccharide was sized by microfluidication before conjugation . The sizes of the saccharides after microfluidization are given in Table 2. Table 1. Specific conditions of activation / coupling / reaction arrest of the conjugates of PS S. pneumoniae-Protein DITTIDTlPhtDIPIy Note: pHa, c, q corresponds to pH for activation, coupling and reaction stop, respectively Characterization Each conjugate was characterized and met the specifications described in table 2. The content of the polysaccharide (μg / ml) was measured by the test of Resorcinol and the protein content (μg / ml) by the Lowry test. The final PS / PD ratio (w / w) was determined by the ratio of the concentrations. Free polysaccharide content (%) The free polysaccharide content of the conjugates was maintained at 4 ° C or stored for 7 days - at 37 ° C, it was determined in the supernatant obtained after incubation with carrier protein antibodies to ammonium sulfate saturated, followed by centrifugation. An a-PS / a-PS ELISA was used for the quantification of the free polysaccharide in the supernatant. The absence of conjugate was also controlled by an a / a-PS carrier protein ELISA. Antigenicity The antigenicity in the same conjugates was analyzed in a sandwich ELISA where the capture and detection of antibodies were a-PS and a-protein, respectively. Free protein content (%) The unconjugated carrier protein can be separated from the conjugate during the purification step. The free residual protein content was determined using size exclusion chromatography (TSK 5000-PWXL) followed by UV detection (214 nm). The allowed elution conditions separate the free carrier protein and the conjugate. The content of free protein in amounts of 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 The molecular weight distribution (Kav) and stability were measurements in a gel filtration of CLAR-SEC (TSK 5000-PWXL) for conjugates maintained at 4 ° C and stored for 7 days at 37 ° C. The characterization of 10/11/13/14 valences is given in the table 2 (see comment below) Protein conjugates can be absorbed into aluminum phosphate and pooled to form the final vaccine.

Conclusion Immunogenetic conjugates have been produced, which have since been shown to be components of a promising vaccine.

Table 2. Characteristics of the conjugates * Size of PS after microfluidization of native PS A 10-valent vaccine was made by mixing the serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugates (for example at a dose of 1, 3, 1, 1, 1, 1, 1, 3, 3, 1 μg of saccharide, respectively per human dose). An 11-valent vaccine was additionally added by adding the conjugate of serotype 3 of Table 5 (for example to 1 μg of saccharide per human dose). A 13-valent vaccine was additionally added by adding the conjugates of serotypes 19A and 22F (with 22F directly linked to PhtD, or alternatively through an ADH linkage) [for example at a dose of 3 μg each of each of saccharides per human dose]. A 14-valent vaccine can be made additionally by adding the serotype 6A conjugate above [for example at a dose of 1 μg of saccharide per human dose].

Example 3: Evidence that the inclusion of Haemphilus influenzae protein D in an immunogenetic composition of the invention can provide enhanced protection against the acute otitis media (AOM). Study design The study used a 11Pn-PD vaccine - comprising serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F each conjugated to protein D of H. influenzae (with reference to the table 5 in example 4). Subjects were randomly selected into two groups to receive four doses of the 11Pn-PD or Havrix vaccine at approximately 3, 4, 5 and 12-15 months of age. All subjects received the Infanrix-hexa vaccine from GSK Biologicals (DTPa-HBV-IPV / Hib) concomitantly at 3, 4 and 5 months of age. Infanrix-hexa is a combination of Pediarix and Hib mixed before administration. The efficacy test for the "Compliant protocol" analysis started 2 weeks after the administration of the third dose of vaccine and continued until 24-27 months of age. The nasopharyngeal vehicle of S. pneumoniae and Hemophilus influenzae was evaluated in a selected subset of subjects. Parents were advised to consult the investigator if their child became sick, had ear pain, spontaneous perforation of the tympanic membrane, or spontaneous discharge from the ear. If the investigator suspects an AOM episode, the child You will be referred immediately to an otolaryngologist specialist (ENT) to confirm the diagnosis. A clinical diagnosis of AOM was based on the visual appearance of the tympanic membrane (ie redness, bulge, loss of reflex to light) or the presence of fluid effusion from the middle ear (as demonstrated by simple or pneumatic otoscopy or by microscopy). In addition, at least two of the following signs or symptoms had to be present: earache, ear discharge, hearing loss, fever, lethargy, irritability, anorexia, nausea, or diarrhea. If the ENT specialist confirmed the clinical diagnosis, a specimen of middle ear fluid is collected by tympanocentesis for the bacteriological test. For subjects with repeat visits with illness, a new episode of AOM was considered to have 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 was different from the previous isolate regardless of the interval between the two consecutive episodes. Results of the trial A total of 4968 children were recruited, 2489 in the 11Pn-PD group and 2479 in the control group. There were no major differences in the demographic characteristics or risk factors between the two groups.

Clinical Episodes and AOM Case Definition During the protocol review period, a total of 333 episodes of clinical AOM were recorded in the 11Pn-PD group and 499 in the control group. Table 3 presents the protective efficacy of the vaccine of 11Pn-PD and both 7-valence vaccines previously tested in Finland (Eskola et al N Engl J Med 2001; 344: 403-409 and Kilpi et al., Clin Infect Dis 2003 37: 1 155-64) against any episode of AOM and AOM caused by different pneumococcal serotypes, H. influenzae, NTHi and M. catarrhails. The statistically significant and clinically relevant reduction of 33.6% of the total burden of AOM disease was achieved with 11Pn-PD, regardless of the etiology (Table 3). The total efficacy against the episodes of AOM due to the pneumococcal serotypes 11 contained in the 11Pn-PD vaccine was 57.6% (Table 3). Another important finding in the current study is the 35.6% protection provided by the 11Pn-PD vaccine against AOM caused by H. influenzae (and specifically 35.3% protection provided by NTHi). This finding is of major clinical importance, given the growing importance of H. influenzae as a major cause of AOM in the pneumococcal conjugate vaccine. In accordance with the protection provided against AOM, the 11Pn-PD vaccine also reduced the nasopharyngeal vehicle of H. influenzae after the booster dose in the second year of life. These findings are contrary to previous observations in Finland where, for both 7-valent pneumococcal conjugate vaccines, an increase in AOM episodes due to H. influenzae was observed (Eskola et al., And Kilpi et al.) As evidence of the etiological replacement. A clear correlation between protection against AOM episodes due to Hi and antibody levels against carrier protein D, could not be established, since post-primate anti-PD IgG antibody concentrations in 11Pn-PD vaccines remained free of the AOM Hi episode were essentially the same as the post-primary IgG anti-PD antibody levels measured in 11Pn-PD vaccines that developed at least one episode of AOM Hi during the period of efficacy testing. However, although no correlation could be established between the biological impact of the vaccine and the immunogenicity of post-primary anti-PD IgG, it is reasonable to assume that the PD carrier protein, which is highly conserved among strains of H. influenzae, has contributed in large part to the induction of protection against Hi. The effect on AOM disease was accompanied by an effect on the nasopharyngeal vehicle that was of similar magnitude for the pneumococci of the vaccine serotype and H. influenzae (figure table 1). This reduction of the nasopharyngeal vehicle of H influenzae in PD conjugate receptors supports the hypothesis of a direct protective effect of the PD conjugate vaccine against H. influenzae, even if the protective efficacy could not be correlated to the immune responses of anti-PD IgG as measured by ELISA. In a following experiment, a model of Chinchilla otitis media was used with serum groups of infants immunized with the 11-valent formulation of this example or with the 10-valent vaccine of Example 2 (see also Table 1 and Table 2 and Table 2). subsequent comments). Both groups induce a significant reduction in the percentage of animals with otitis media against the pre-immune serum group. There is no significant difference between the immune groups of 10 and 11 valences. This shows that both vaccines have a similar potential to induce protection against the otitis media caused by H. influenzae not classifiable in this model.

Table 3 VO fifteen pneumococcal vaccine: for 11 Pn-PD = 11 serotypes, for Prevnar and 7v-0MP = 7 serotypes MEF = middle ear fluid Example 4 Selection of carrier protein for serotype 19F Analysis of ELISA used The ELISA method of 22F inhibition was essentially based 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, the purified pneumococcal polysaccharides were mixed with human serum albumin methylated and absorbed in Nunc Maxisorp ™ high binding microtitre plates (Roskilde, DK) overnight at 4 ° C. Plates were blocked with 10% fetal calf serum (FBS) in PBS for 1 hour at room temperature with shaking. The 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 were also diluted in microtiter plates with the same shock absorber. An internal reference calibrated against the standard 89-SF serum using the serotype-specific IgG concentrations in 89-SF, was treated in the same manner and was included in each plate. After washing, bound antibodies were detected using the anti-human IgG monoclonal antibody conjugated with peroxidase (Stratech Scientific Ltd, Soham, UK) were diluted in 10% FBS (in PBS), and incubated for 1 hour at room temperature with agitation. The color was developed using a ready-to-use substrate kit Single component tetramethylbenzidine peroxidase enzyme immunoassay (BioRad, Hercules, CA, US) in dark at room temperature. The reaction was stopped with 0.18 M H2SO4, and the optical density was read at 450 nm. Serotype-specific IgG concentrations (at μg / ml) in the samples were calculated by referring to optical density points within the defined limits of the internal reference serum curve, which is modeled by a 4-parameter logistic equation calculated with SoftMax Pro ™ software (Molecular Devices, Sunnyvale, CA). The limit for ELISA was 0.05 μg / ml IgG for all serotypes that considered the limit of detection and the limit of quantification. Analysis of opsonophagocytosis At the WHO consultation meeting in June 2003, it was recommended to use an OPA analysis as required in Romero-Steiner et al. Clin Diagn Lab Immunol 2003 10 (6) pp1019-1024. This protocol was used to test the OPA activity of the serotypes in the following tests. Preparation of conjugates In the studies 11 Pn-PD &Di-001 and 11 Pn-PD &Di-007, three 11-valent vaccine formulations were included (Table 4) in which 3 μg of polysaccharide 19F was conjugated to the diphtheria toxoid (19F-DT) instead of 1 μg polysaccharide conjugated to protein D (PD 19F-). The conjugation parameters for the 11Pn-PD, 11 Pn-PD & Di-001 and 11 Pn-PD & Di-007 studies are they are described in tables 5, 6 and 7 respectively. Anti-pneumococcal antibody responses and OPA activity against 19F serotypes one month after primary vaccination with these 19F-DT formulations are shown in Table 8 and 9, respectively. Table 10 shows the concentrations of the 22F-ELISA antibody and the percentages of subjects who reached the threshold of 0.2 μg / ml before and after the 23-valent simple polysaccharide booster vaccination. Opsonophagocytic activity showed clear improvement for the antibodies induced with these 19F-DT formulations as demonstrated by the highest seropositivity indices (opsontophagocytic titers> 1: 8) and OPA GMTs one month after primary vaccination (Table 9 ). One month after the 23-valent simple polysaccharide booster vaccination, the opsonophagocytic activity of the 19F antibodies remained significantly better for children pre-injected with the 19F-DT formulations (Table 11). Table 12 presents the immunogenicity data after a booster dose of 11Pn-PD in children previously treated with the 19F-DT or 19F-PD conjugates compared to a fourth consecutive dose of Prevnar®. Due to the cases of progress reported after the introduction of Prevnar® in the United States of North America, the improved opsonophagocytic activity against the 19F serotypes when conjugated to the DT carrier protein may be an advantage for the candidate vaccine. Table 13 provides the ELISA and OPA data for the 19F-DT conjugate with respect to the 19A cross reaction serotype. It was found that 19F-DT induces little OPA activity but significant against 19A.

Table 4. Pneumococcal conjugate vaccine formulations used in clinical studies.

Table 5. Specific conditions of activation / coupling / reaction arrest of the conjugates of PS S. pneumoniae-Protein DITTIDT / PhtDIPIy Table 6. Specific activation / coupling / reaction arrest conditions of the PS S. pneumoniae-DIDT protein conjugates for the 11Pn-PD & Di-001 study Table 7. Specific conditions for activation of the reaction arrest of the conjugates of PS S. pneumoniae-Protein DIDT for the study 11Pn-PD &Di-007 Table 8. Percentage of subjects with 19F antibody concentration and geometric average antibody concentrations of antibody 19F > _ 0.02 μglml (GMCs with 95% Cl; μglml) one month after the primary vaccination of 1 μg 19F-PD, 3 μg 19F-DT or Prevnar (2 μg 19F-CRM) (total cohort) The composition of various compositions is given in Table 4.

Table 9. Percentage of subjects with a title of 19F OPA > 1: 8 and GMTs of 19F OPA one month after primary vaccination with 1 μg 19F-PD, 3 μg 19F-DT or Prevnar (2 μg 19F-CRM) (total cohort) The composition of various compositions is given in table 4.

Table 10. Percentage of subjects with antibody concentration 19F = 0.20 μglml and 19F antibody GMCs (μglml) before and one m is after reinforcement of 23-valent simple polysaccharide in children previously treated with 1 μg 19F-PD, 3 μg 19F-DT or Prevnar (2 μg 19F-CRM) (total cohort) The composition of different compositions is given in table 4.

Table 11. Percentage of subjects with a title of 19F OPA > .1: 8 and GMTs of 19F OPA before and one month after the 23-valent simple polysaccharide reinforcement in children previously treated with 1 μg 19F-PD, 3 μg 19F-DT or Prevnar (2 μg 19F-CRM) ( total cohort) The composition of different compositions is given in Table 4. Table 12. Percentage of subjects with antibody concentrations > 0.2 μglml, OPA > 1: 8 and GMCsIGMT against pneumococci 19F a m is after the reinforcement of 1 1 Pn-PD or Prevnar in children previously treated with 1 μg 19F-PD, 3μg 19F-D T or Prevnar (2 μg 19F-CRM) (total cohort) The position of different compositions is given in table 4.

Table 13. Percentage of subjects with antibody concentrations > 0.2 μglml, OPA > 1: 8 and GMCsIGMT against 19A pneumococci one month after primary vaccination with 1 μg 19F-PD, 3μg 19F-DT or Prevnar (2 μg 19F-CRM) (total cohort) The composition of different compositions is given in Table 4. Example 5: Adjuvant experiments in pre-clinical models: impact on the immunogenicity of 11-valent pneumococcal polysaccharide conjugates in elderly Rhesus monkeys To optimize the response produced for 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 on days 0 and 28 with 500 μl of PS conjugates of 11 valencias absorbed in 315 μg of AIPO4 or conjugates of PS of 11 valences mixed with adjuvant C. In both vaccine formulations, each 11-valent PS conjugate was 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 dose of the human dose of vaccine (5 μg of each saccharide per human dose except 6B [10 μg]) conjugated according to the conditions in Table 6 (example 4), except that 19F is made according to the following CDAP process conditions: saccharide sized at 9 mg / ml, PD at 5 mg / ml, an initial ratio of PD / PS of 1.2 / 1, a CDAP concentration of 0.75 mg / mg PS, pHa = pHc = pH 9.0 / 9.0 / 90 and a coupling time of 60 mM.

Anti-PS ELISA IgG levels and opsonophagocytosis titers were dosed in serum collected on day 42.

The B-cell frequencies of anti-PS3 memory were measured by Elispot of the peripheral red blood cells collected on day 42. According to the results shown below, adjuvant C significantly improved the immunogenicity of the 11-valent PS conjugates against the conjugated with AIPO4 in elderly monkeys. The new adjuvant improved the IgG responses to PS (table 1) and the opsonophagocytosis antibody titers (table 14). There was also evidence of support that the frequency of memory B cells of PS3 specific is increased by the use of adjuvant C (table 2). Table 14. Immunogenicity of conjugate in elderly Rhesus monkeys (titers after opsonophagocytosis II) B cell elispot The principle of the analysis is based on the fact that memory B cells mature in plasma cells in vitro after cultivation with CpG for 5 days. The antigen-specific plasma cells generated in vitro can be easily detected and therefore used in the B cell Elispot analysis. The number of specific plasma cells reflects the frequency of the memory cells at the start of the client. Briefly, plasma cells generated in vitro are incubated in culture dishes coated with antigen. Antigen-specific plasma cells form antibody / antigen sites, which are detected by the conventional immunoenzyme method and are used as B-cells. memory. In the current study, polysaccharides have been used to cover culture plates to list the respective memory B cells. The results are expressed as the frequency of memory-specific B cells to PS within one million memory B cells. The study shows that adjuvant C may be able to solve the known problem of PS3 increase capacity (see 5th International Symposium on Pneumococcal and Pneumococcal Diseases, 2-6 April 2006, Alice Springs, Central Australia. to serotype 3 pneumococcal conjugate, Schuerman L, Prymula R, Poolman J. Abstract book p 245, PO10.06). Example 6. Efficacy of detoxified pneumolysin (dPly) as a protein carrier to improve the immunogenicity of PS 19F in young Balb / c mice. Groups of 40 female Balb / c mice (4 weeks old) were IM immunized on days 0, 14 and 28 with 50 μl of 4-valent simple PS or PS conjugated with 4-valent dLli, 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 ELISA IgG levels were dosed in sera collected on day 42. The anti-PS19F response, shown as an example in Table 3, was strongly improved in mice treated with dPli conjugates of 4. valences compared to mice immunized with simple PS. The same improvement was observed for the responses of anti-PS8, 12F and 22F IgG (data not shown). Example 7. Effectiveness of histidine triad pneumococcal D protein (PhtD) as a protein carrier to enhance the immunogenicity of PS 22F in young Balb / c mice. Groups of 40 female Balb / c mice (4 weeks old) were immunized IM on days 0, 14 and 28 with 50 μl of simple 4-valent PS or PS conjugated with 4-valent PhtD, both mixed with adjuvant C. Both formulations of vaccine were composed of 0.1 • μg (amount of saccharide) of each of the following PS: PS8, PS12F, PS19F and PS22F. IgG levels of anti-PS ELISA were dosed in sera collected on day 42. The anti-PS22F response, shown as an example in Table 4, was strongly improved in mice treated with 4-valent PhtD conjugates compared to mice immunized with simple PS. The same improvement was observed for the responses of anti-PS8, 12F and 19F IgG (data not shown).

Example 8. Immunogenicity in 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 11-valent PS conjugates or 13-valent PS conjugates, both mixed with adjuvant C (see down). The valence vaccine formulation 11 was composed of 0. 1 μg 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 the commentary regarding the 11-valent vaccine discussed under Table 2). The vaccine formulation contained 13 valencies in addition to 0.1 μg of conjugates of PS19A-dPly and PS22F-PhtD (see table 1 and the commentary regarding the 13-valent vaccine discussed under table 2 [using the 22F conjugate directly] ). In group 2 and 4 the pneumolysin carrier was detoxified with treatment of GMBS, in group 3 and 5 was made with formaldehyde. In groups 2 and 3 PhtD was used to conjugate PS 22F, in groups 4 and 5 a fusion of PhtD_E (construct VP147 of WO 03/054007) was used. In group 6 19A, diphtheria toxoid and 22F were conjugated to protein D. Anti-PS19A and 22F ELISA IgG levels were dosed in individual sera collected on day 42. The ELISA IgG response generated to another PS was measured in grouped sera. 19A-dPly and 22F-PhtD administered within the formulation of 13-valent conjugate vaccine were shown to be immunogenic in elderly C57BI mice (table 15). The immune response induced against the other PS was not adversely affected in the mice treated with the 1 3-valent formulation compared to those immunized with the 1-valent formula.

Table 15. Immunogenicity of PS in elderly C57BI mice (IgG levels after lll) 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 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 the commentary regarding the 11-valent vaccine discussed below in Table 2). The 13-valent vaccine formulation also contained 0.1 μg of PS19A-dPly and PS22F-PhtD conjugates (see Table 1 and the commentary regarding the 13-valent vaccine discussed below in Table 2 [using 22F directly conjugated]) . In group 2 and 4 the pneumolysin carrier was detoxified with GMBS treatment, in group 3 and 5 it was made with formaldehyde. In groups 2 and 3 PhtD was used to conjugate PS 22F, in groups 4 and 5 a fusion of PhtD_E was used (construct VP 147 of WO 03/054007). In group 6 19A it was conjugated to diphtheria toxoid and 22F to protein D. Anti-PS19A and IgG ELISA levels of 22F were dosed in individual sera collected on day 42.

IgG response of ELI SA generated to the other PS was measured in the pooled sera. 19A-d Ply and 22 F-PM D administered within the 13-valent conjugate vaccine formulation were determined immunogenetic in young Balb / c mice (Table 16). The immune response induced against the other PS was not adversely affected in the mice treated with the formulations of 1 3-valent compared to those immunized with the 1-valent formulation. Table 16. Immunogenicity of PS in young Balb / c mice (IgG levels after lll) Table 17. Immunogenicity of PS in young Balb / c mice (IgG levels after lll) t Example 10. Immunogenicity in guinea pigs of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD Groups of 20 young guinea pigs (Hartley breed, 5 weeks of age), were immunized IM on days 0, 14 and 28 with 125 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 0.25 μg 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 the commentary regarding the 11-valent vaccine discussed * below table 2). The 13-valent vaccine formulation also contained 0.1 μg of PS19A-dPly and PS22F-PhtD conjugates (see Table 1 and comment with respect to the 13-valent vaccine discussed below in Table 2 [using 22F directly conjugated] ). In group 2 and 4, the pneumolysin carrier was detoxified with GMBS treatment, in group 3 and 5 it was made with formaldehyde. In groups 2 and 3 PhtD was used to conjugate PS 22F, in groups 4 and 5 a fusion of PhtD_E was used (construct VP 147 of WO 03/054007). In group 6 19A was conjugated to diphtheria toxoid and 22F to protein D. Anti-PS19A and 22G ELISA levels of 22F were dosed in individual sera collected on day 42. The ELISA IgG response generated to the other PS It was measured in grouped sera.

Example 11. Formulations made and tested a) The following formulations are made (using the 13-valent vaccine from Table 1 and serotype 3 from Table 5 - see the comment regarding the 14-valent vaccine below Table 2 [using 22F conjugated directly or through an ADH link]). The saccharides are formulated with aluminum phosphate and 3D-MPL as shown below. b) The same saccharide formulation with each of the following adjuvants: The concentration of the emulsion components per 500 μl dose is shown in the following table. Ingredients Adjuvant Adjuvant Adjuvant A1 250 μl A2 125 μl A3 o / po / p 50 μl o / p emulsion emulsion emulsion Tocopherol 11.88 mg 5.94 mg 2.38 mg alfa squalene 10.7 mg 5.35 mg 2.14 mg Tween 80 4.85 mg 2.43 mg 0.97 mg Ingredients Adjuvant Adjuvant Adjuvant Adjuvant A4 A5 A6 A7 250 μl o / w 250 μl o / w 125 μl o / w 50 μl o / w emulsion emulsion emulsion emulsion Tocopherol 11.88 mg 11.88 mg 5.94mg 2.38mg alpha squalene 10.7 mg 10.7 mg 5.35 mg 2.14mg Tween 80 4.85 mg 4.85 mg 2.43mg 0.97mg 3D-MPL 50 μg 25 μg 25 μg 10 μg c) Saccharides are also formulated with two liposome-based adjuvants: Composition of adjuvant B1 Qualitative quantitative (per dose of 0.5 ml) - DOPC 1 mg - Cholesterol 0.25 mg 3D-MPL 50 μg QS21 50 μg KH2PO 1 3.124 mg buffer Na2HPO? 0.290 mg NaCI 2,922 mg buffer (100 mM) WFI is. add 0.5 ml solvent pH 6.1 • 1. total concentration of PO = 50 mM Composition of adjuvant B2 Qualitative Quantitative (per dose of 0.5 ml) Liposomes: - DOPC 0.5 mg - cholesterol 0.125 mg 3D-MPL 25 μg QS21 25 μg KH2PO41 3.124 mg buffer Na2HPO4? 0.290 mg buffer NaCI 2.922 mg (100 mM) WFI e s. added to 0.5 ml solvent pH 6.1 d) The saccharides are also formulated with adjuvant C (see above for other compositions where this adjuvant has been used): Qualitative Quantitative (per dose of 0.5 ml) Oil in water emulsion: 50 μl squalene 2.136 mg - tocopherol at 2.372 mg Tween 800.97 mg cholesterol 0.1 mg 3D-MPL 50 μg QS21 50 μg KH2PO41 0.470 mg buffer Na2HPO? 0.219 mg NaCI buffer 4.003 mg (137 mM) KCl 0.101 mg (2.7 mM) WFI is. added to 0.5 ml solvent pH 6.8 Example 12. Impact of conjugate chemistry on the immunogenicity of the 22F-PhtD conjugate in Balb / c mice The groups of 30 female Balb / c mice were immunized by the intramuscular route 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 saccharides / PS for PS 4, 18C, 19A, 19F and 22F and 0.1 μg saccharides / PS for the other PS). PS 18C was conjugated to tetanus toxoid, 19F to diphtheria toxoid, 19A to PIy detoxified with formaldehyde, 22F to PhtD and the other PS to PD. Two formulations, consisting of 22F-PhtD prepared by direct chemistry of CDAP or 22F-AH-PhtD (PS derived from ADH), were compared. See example 2, table 1 and the comment below in table 2 for the characteristics of the 13-valent vaccine made with 22F conjugated directly or via an ADH separator. The vaccine formulations were supplemented with adjuvant C. IgG levels of anti-PS22F ELISA and opsonophagocytosis titres were measured in sera collected on day 42. 22F-AH-PhtD was shown much more immunogenic than 22F-PhtD in terms of both IgG levels (Table 5) and opsonophagocytosis titles (Table 6). Example 13. Impact of new adjuvants on the immunogenicity of capsular PS conjugates of Streptoccoccus pneumoniae Groups of 40 female Balb / c mice were immunized by the IM route 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 / PS for PS 4, 18C, 19A, 19F and 22F and 0.1 μg / PS for the other PS). PS 18C was conjugated to tetanus toxoid, 19F to diphtheria toxoid, 19A to PIy detoxified with formaldehyde, 22F to PhtD and the other PS to PD. See example 2, table 1 and the comment below from table 2 for the characteristics of the 13-valent vaccine made with 22F conjugated directly. Four formulations were compared, complemented with AIPO, adjuvant A1, adjuvant A4 or adjuvant A5. The anti-PS, PIy, PhtD and IgG ELISA levels of PD were measured in the sera collected on day 42 and collected by group. The following ratio was calculated for each antigen: The level of IgG induced with the new adjuvant tested / level of IgG induced with AIPO4. All the new adjuvants tested improved at least 2 times the immune responses to 13-valent conjugates compared to the classic AIPO formulation (table 7) - Example 14. Protective effectiveness of a detoxified PhtD / Ply kit in a mono-pneumonia model pneumococcal The groups of 6 Rhesus monkeys (aged 3 to 8 years), selected as having the lowest pre-existing levels of anti-19F antibody, were immunized intramuscularly on days 0 and 28 with 11-valent PS conjugate conjugates (ie 1 μg of PS 1, 3, 5, 6B, 7F, 9V, 14 and 23F, and 3 μg of PS 4, 18C and 19F) or PhtD (10 μg) + PIy detoxified with formaldehyde (10 μg) or only the adjuvant. PS 18C was conjugated to tetanus toxoid, 19F to diphtheria toxoid and the other PS to PD. See example 2, table 1 and the comment below in table 2 for the characteristics of the 11-valent vaccine. All the formulations were complemented with adjuvant C. The pneumococci type 19F (5,108 cfu) were inoculated in the right lung on day 42. The colonies were counted in the bronchoalveolar lavages collected on days 1, 3 and 7 after the challenge. The results were expressed as the number of animals per group even if they died, the lung was colonized or purified on day 7 after the challenge. As shown in Figure 8, good protection close to statistical significance (despite the low number of animals used), was obtained with 11-valent conjugates and the set of PhtD + dPly (p <0.12, Fisher test Exact) in comparison to the group of only the adjuvant. Example 15. Impact of conjugation chemistry on anti-PhtD antibody response and protective efficacy against a type 4 challenge induced by 22F-PhtD conjugates Groups of 20 female OF1 mice were immunized by the intramuscular route 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 only the adjuvant. Both monovalent 22F conjugates were made by the example processes 2 (see also Table 1 and Table 2). Each formulation was supplemented with adjuvant C. The IgG levels of EL I SA of antis-PhtD were measured in the sera collected on day 27. The mice were challenged intranasally with 5,106 cfu of type 4 pneumococci on day 28. (ie a pneumococcal serotype potentially not covered by the PS present in the vaccine formulation tested). Induced mortality was monitored until day 8 after the challenge. 22 F-AH-PM D induced a significantly higher anti-PhtD IgG response and better protection against type 4 challenge than * 22F-PhtD.

Claims (132)

1. An immunogenic composition of Streptococcus pneumoniae comprising 9 or more, 10 or more, 11 or more, 13 or more, or 14 or more capsular saccharides of different serotypes of S. pneumoniae conjugated to 2 or more different carrier proteins, wherein the composition comprises the capsular saccharide of the serotype 19F conjugated to the diphtheria toxoid or CRM197, optionally wherein 19F is the only one taking out in the conjugate composition the diphtheria toxoid or CRM197.

2. An immunogenetic composition according to claim 1, wherein the 19F serotype is conjugated to diphtheria toxoid.

3. An immunogenetic composition according to claim 1 or 2, wherein the composition further comprises protein D of Hemophilus influenzae.

4. An immunogenetic composition according to any preceding claim, wherein the 19F capsular saccharide is conjugated directly to the carrier protein.

5. An immunogenetic composition according to any of claims 1 to 3, wherein the 19F capsular saccharide is conjugated to the carrier protein via a bond.

6. An immunogenetic composition according to claim 5, wherein the bond is bifunctional.

7. The immunogenetic composition of claim 5 or 6, where the link is ADH.

The immunogenetic composition of claims 5, 6, or 7, wherein the bond is linked to the carrier protein by carbodiimide chemistry, preferably using EDAC.

The immunogenetic composition of any of claims 5 to 8, wherein the saccharide is conjugated to the link before the carrier protein is conjugated to the linkage.

The immunogenetic composition of any of claims 5 to 8, wherein the carrier protein is conjugated to the link before the saccharide is conjugated to the linkage.

11. The immunogenetic composition of any preceding claim, wherein the 19F saccharide is conjugated to the carrier protein or linkage using CDAP chemistry.

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

The immunogenetic composition of any preceding claim, wherein the average size (for example Mw) of the 19F saccharide is above 100 kDa.

The immunogenetic composition of claim 13, wherein the average size (for example Mw) of the 19F saccharide is between 100-750, 110-500, 120-250, or 125 and 150 kDa.

15. The immunogenetic composition of claim 13 or 14, wherein the 19F saccharide is a native polysaccharide or is dimensioned by a factor of no more than x5.

16. The immunogenetic composition of claim 13, 14 or 15, wherein the 19F saccharide has been sized by microfluidization.

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

18. An immunogenetic composition according to any preceding claim, wherein at least 8 of the capsular saccharides are conjugated to the same carrier protein.

19. An immunogenetic composition according to claim 18, wherein the carrier protein is not diphtheria toxoid and / or is not CRM197.

20. An immunogenetic composition according to any preceding claim comprising 2 different carrier proteins.

21. An immunogenetic composition according to any preceding claim, comprising 3, 4, 5 or 6 different carrier proteins.

22. An immunogenetic composition according to any preceding claim, wherein one or more or all of the carrier proteins are selected from the group consisting of DT, CRM 197, TT, fragment C, dPly, PhtA, PhyB, PhtD, PhtE, PhtDE OmpC, PorB and protein D of Hemophilus influenzae.

23. An immunogenetic composition according to claim 22, wherein a carrier protein is the D protein.

24. An immunogenetic composition according to claim 22, wherein a carrier protein is TT.

25. An immunogenetic composition according to any of claims 22, 23 or 24, wherein TT and protein D are present as carrier proteins.

26. A immunogenetic composition according to any of claims 22, 23, 24 or 25, wherein at least 8 of the capsular saccharides are conjugated to protein D.

27. An immunogenetic composition according to any preceding claim, in wherein the composition comprises capsular saccharide 18C conjugated to TT, optionally wherein 18C is the only saccharide in the composition conjugated to tetanus toxoid.

28. An immunogenetic composition according to any preceding claim, wherein the capsular saccharide 18C is conjugated directly to the carrier protein.

29. An immunogenetic composition according to any of claims 1 to 27, wherein the 18C capsular saccharide is conjugated to the carrier protein via a bond.

30. An immunogenetic composition according to claim 29, wherein the bond is bifunctional.

31. The immunogenetic composition of claim 29 or 30, wherein the linkage is ADH.

32. The immunogenetic composition of claims 29, 30, or 31, wherein the bond is bound to the carrier protein by carbodiimide chemistry, preferably using EDAC.

33. The immunogenetic composition of any of claims 29 to 31c, wherein the saccharide is conjugated to the link before the carrier protein is conjugated to the linkage.

34. The immunogenetic composition of any of claims 29 to 31, wherein the carrier protein is conjugated to the link before the saccharide is conjugated to the link.

35. The immunogenetic composition of any preceding claim, wherein the 18C saccharide is conjugated to the carrier protein or linkage using CDAP chemistry.

36. The immunogenetic composition of any preceding claim, wherein the 18C saccharide is conjugated to the carrier protein or the linkage using reductive amination.

37. The immunogenetic composition of any preceding claim, wherein the ratio of the carrier protein to the 18C saccharide is between 0.5: 1-5: 1, 1: 1-4: 1,

1. 5: 1-3: 1, or 2: 1 and 2.5: 1 (p / p).

38. The immunogenetic composition of any preceding claim, wherein the average size (for example Mw) of the saccharide of 18C is above 50 kDa.

39. The immunogenetic composition of the claim 38, wherein the average size (for example Mw) of the 18C saccharide is between 50-500, 60-400, 70-300, 75-200, or 80 and 100 kDa.

40. The immunogenetic composition of claim 39, wherein the 18C saccharide is a native polysaccharide or is sized by a factor of not more than x5.

41. The immunogenetic composition of claim 38, 39 or 40, wherein the saccharide of 18C has been sized by microfluidization.

42. The immunogenetic composition of any preceding claim, wherein the dose of saccharide conjugate of 18C is between 1 and 10 μg, 1 and 5 μg, or 1 and 3 μg of saccharide

43. The immunogenetic composition of claim 42, in wherein the dose of the saccharide conjugate of 18C is 3 μg of the saccharide.

44. An immunogenetic composition according to any preceding claim, wherein a capsular saccharide conjugate of serotype 6B is present, but is not conjugated to DT and / or to CRM197.

45. An immunogenetic composition according to any preceding claim, wherein a capsular saccharide conjugate of serotypes 23F is present, but is not conjugated to DT and / or to CRM197.

46. An immunogenetic composition according to any preceding claim, wherein at least serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F are present as conjugated saccharides.

47. An immunogenic composition according to any preceding claim, wherein serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F are present and all are conjugated to protein D.

48. An immunogenetic composition according to to any preceding claim, which additionally comprises serotype 3 present as conjugated saccharide.

49. An immunogenetic composition according to claim 48, wherein serotype 3 is conjugated to protein D.

50. An immunogenetic composition according to any preceding claim, which additionally comprises (conjugated capsular saccharide of) serotype 6A and / or 15B.

51. An immunogenetic composition according to any preceding claim, which additionally comprises (conjugated capsular saccharide of) serotypes 19A.

52. An immunogenetic composition according to any preceding claim, which additionally comprises (conjugated capsular saccharide of) serotype 22F.

53. An immunogenetic composition according to any preceding claim, which additionally comprises (capsular saccharide conjugated of) serotypes 8.

54. An immunogenetic composition according to any preceding claim which additionally comprises (conjugated capsular saccharide of) serotype 12F.

55. An immunogenetic composition according to any preceding claim, wherein at least one of the capsular saccharides is conjugated directly to the carrier protein.

56. An immunogenetic composition according to any preceding claim, wherein at least one of the capsular saccharides is conjugated to the carrier protein via a bond.

57. An immunogenetic composition according to claim 56, wherein the bond is bifunctional.

58. The immunogenetic composition of claim 56 or 57, wherein the linkage is ADH.

59. The immunogenetic composition of claims 56, 57, or 58, wherein the linkage is bound to the carrier protein by carbodiimide chemistry, preferably using EDAC.

60. The immunogenetic composition of any of claims 56 to 59, wherein the saccharide is conjugated to the link before the carrier protein is conjugated to the linkage.

61. The immunogenetic composition of any of claims 56 to 59, wherein the carrier protein is conjugated to the link before the saccharide is conjugated to the link.

62. The immunogenetic composition of any previous claim, wherein at least one of the capsular saccharides is conjugated to the carrier protein and / or to the binding using CDAP chemistry.

63. The immunogenetic composition of any preceding claim, wherein at least one of the capsular saccharides is conjugated to the carrier protein and / or the linkage using reductive amination.

64. The immunogenetic composition of claim 63, wherein the capsular saccharide of 3 is conjugated to the carrier protein and / or to the linkage using reductive amination.

65. The immunogenetic composition of claim 63 or 64, wherein the capsular saccharide of 1 is conjugated to the carrier protein and / or the linkage using reductive amination.

66. The immunogenetic composition of any preceding claim, wherein the ratio of the carrier protein to the saccharides is between 0.5: 1-5: 1, 1: 2 and 2.5: 1, 1: 1-3.5: 1, or 1.3: 1-3.0: 1.

67. The immunogenetic composition of any preceding claim, wherein the average size (for example Mw) of the saccharides (in the saccharide conjugates) is above 50 kDa, for example 50-1600, 80-1400, 100-1000 , 150-500, or 200-400 kDa.

68. The immunogenetic composition of any preceding claim, comprising serotype 1 (saccharide conjugate) having an average saccharide size (per example Mw) of between 100-100O, 200-800, 250-600, or 300 and 400 kDa.

69. The immunogenetic composition of any preceding claim, comprising 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

70. The immunogenetic composition of any preceding claim, comprising 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

71. The immunogenetic composition of any preceding claim comprising serotype 6B (saccharide conjugate) having an average saccharide size (for example Mw) of between 500-1600, 750-1500, or 1000 and 1400 kDa.

72. The immunogenetic composition of any preceding claim, comprising serotype 7F (saccharide conjugate) having an average saccharide size (eg, Mw) of between 50-1000, 100-750, 150-500, or 200 and 300 kDa

73. The immunogenetic composition of any preceding claim, comprising serotype 9V (saccharide conjugate) having an average saccharide (e.g., Mw) size of between 50-1000, 100-750, 150-500, 200-400, or 250 and 300 kDa.

74. The immunogenetic composition of any previous claim, which comprises 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.

75. The immunogenetic composition of any preceding claim comprising serotype 23F (saccharide conjugate) having an average saccharide size (for example Mw) of between 500-1500, 700-1300, 800-1100, or 900 and 1000 kDa .

76. The immunogenetic composition of any preceding claim, comprising serotype 19A (saccharide conjugate) having an average saccharide size (for example Mw) of between 50 and 800 kDa, 110 and 700 kDa, 110-300, 120- 200, 130-180, or 140-160 kDa.

77. The immunogenetic composition of any preceding claim, comprising serotypes 22F (saccharide conjugate) having an average saccharide size (for example Mw) of between 50 and 800 kDa, 110 and 700 kDa, 110-300, 120- 200, 130-180, or 150-170 kDa.

78. The immunogenetic composition of any preceding claim, comprising serotype 6A (saccharide conjugate) having an average saccharide (e.g. Mw) size of between 500 and 1600 kDa, or 1100 and 1540 kDa.

79. The immunogenetic composition of any previous claim comprising serotype 3 (saccharide conjugate) having an average saccharide size (for example Mw) of between 50 and 1000 kDa, 60 and 800, 70 and 600, 80 and 400, 100 and 300, or 150 and 250 kDa.

80. The immunogenetic composition of any preceding claim, comprising serotypes 5, 6B and 23F as native saccharides.

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

82. The immunogenetic composition of any preceding claim, comprising the conjugates of serotypes 4, 18C and 19F at dosages of 3 μg of saccharide per conjugate.

83. The immunogenetic composition of any preceding claim comprising the conjugates of serotypes 1, 5, 6B, 7F, 9V, 14 and 23F at dosages of 1 μg of saccharide per conjugate.

84. The immunogenetic composition of any preceding claim, which comprises the conjugates of serotypes 6A and / or 3 at dosages of 1 μg of saccharide per conjugate.

85. The immunogenetic composition of any preceding claim, comprising the conjugates of the serotypes 19A and / or 22F at dosages of 3 μg of saccharide per conjugate.

86. The immunogenetic composition of any preceding claim, which additionally comprises the saccharides of unconjugated S. pneumoniae of different serotypes of the conjugates, such that the number of conjugated and unconjugated saccharide serotypes is less than or equal to 23.

87. The immunogenetic composition of any preceding claim, which additionally comprises one or more unconjugated or conjugated S. pneumoniae proteins.

88. The immunogenetic composition of claim 87, comprising one or more non-conjugated S. pneumoniae proteins 89. The immunogenetic composition of claim 87 or 88, wherein one or more S. pneumoniae proteins are selected from the family of polyhistidine triad (PhtX), choline binding protein family (CbpX), truncated CbpX, LytX family, truncated LytX, truncated LytX-truncated CbpX chimeric proteins, detoxified pneumolysin (PIy), PspA, PsaA, Sp128 , Sp101, Sp130, Sp125 and Sp133. 90. The immunogenetic composition of claims 87, 88 or 89, comprising pneumolysin. 91. The immunogenetic composition of any of claims 87 to 90, comprising a PhtX protein. 92. An immunogenetic composition according to any preceding claim, comprising pneumolysin as a free or carrier protein. 93. An immunogenetic composition according to any preceding claim, comprising a PhtX protein as free or carrier protein. 94. The immunogenetic composition of claim 93, wherein the PhtX protein is PhtD or a fusion protein of PhtBD or PhtDE. 95. An immunogenetic composition according to any preceding claim, which additionally comprises an adjuvant. 96. An immunogenetic composition according to claim 95, wherein the adjuvant is a preferential inducer of a Th1 response. 97. An immunogenetic composition according to claim 95 or 96, wherein the adjuvant comprises one or more components selected from QS21, MPL® or an immunoestimulatory oligonucleotide. 98. An immunogenetic composition according to any of claims 95 to 97, wherein the adjuvant comprises QS21 and MPL. 99. The immunogenetic composition of claim 95, wherein the adjuvant comprises a liposome carrier. 100. The immunogenetic composition of claim 99, wherein the adjuvant comprises (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). 101. The immunogenetic composition of claim 99 or 100, wherein the adjuvant comprises (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) sterol (for example cholesterol). 102. The immunogenetic composition of claims 99-101, wherein the adjuvant comprises (per 0.5 ml dose) 5-60, 10-50, or 20-30 μg (eg 5-15, 40-50, 10, 20, 30, 40 or 50 μg) derived from lipid A (for example 3D-MPL). 103. The immunogenic composition of claims 99-102, wherein the adjuvant comprises (per 0.5 ml dose) 5-60, 10-50, or 20-30 μg (eg 5-15, 40-50, 10, 20, 30, 40 or 50 μg) saponin (for example QS21). 104. The immunogenetic composition of claim 95, wherein the adjuvant comprises an oil / water emulsion. 105. The immunogenetic composition of claim 104, wherein the adjuvant comprises (per dose of 0.5 ml)

0. 5-15, 1-13, 2-11, 4-8, or 5-6 mg (for example 2-3, 5-6, or 10-11 mg) metabolizable oil (such as squalene). 106. The immunogenetic composition of claim 104 or 105, wherein the adjuvant comprises (per 0.5 ml dose) 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) emulsifiers (such as Tween 80). 107. The immunogenetic composition of claims 104-106, wherein the adjuvant comprises (per 0.5 ml dose) 0.5-20, 1-15, 2-12, 4-10, 5-7 mg (e.g. 11-13 , 5-6, or 2-3 mg) tocol (such as alpha tocopherol). 108. The immunogenetic composition of claims 104-107, wherein the adjuvant comprises (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) derived from lipid A (for example 3D-MPL). 109. The immunogenetic composition of claims 104-108, wherein the adjuvant comprises (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 (eg 0.2- 0.3, 0.1-0.15, 0.25 or 0.125 mg) sterols (for example cholesterol). 110. The immunogenetic composition of claims 104-109, wherein the adjuvant comprises (per dose of 0.5 ml) 5-60, 10-50, or 20-30 μg (eg 5-15, 40-50, 10, 20, 30, 40 or 50 μg) saponin (for example QS21) 111. The immunogenetic composition of claim 95, wherein the adjuvant comprises a metal salt and a lipid derivative A. 112. The immunogenetic composition of claim 111 , wherein the adjuvant comprises (per dose of 0.5 ml) 100-750, 200-500, or 300-400 μg Al as aluminum phosphate. 113. The immunogenetic composition of the claim 111 or 112, wherein the adjuvant comprises (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) ) lipid A derivative (e.g. 3D-MPL) 114. A vaccine kits, comprising an immunogenetic composition according to any of claims 1 to 94 and additionally comprising for the concomitant or sequential administration an adjuvant as defined in any of claims 96 to 113. 115. A vaccine comprising the immunogenetic composition of any of claims 1 to 113 and a pharmaceutically acceptable excipient. 116. A process for making the vaccine according to claim 115, comprising the step of mixing the immunogenetic composition of any of claims 1 to 113 with a pharmaceutically acceptable excipient 117. A method for immunizing a human host against the disease caused by the infection of Streptococcus pneumoniae, which comprises administering to the host an immunoprotective dose of the immunogenic composition of any of claims 1 to 113 or of the vaccine of claim 115. The method of claim 117, wherein the host Human is an old man, and the disease is either or both of pneumonia or invasive pneumococcal disease. 119. The method of claim 117 or 118, wherein the Human host is an elderly person, and the disease is exacerbations of chronic obstructive pulmonary disease (COPD). 120. The method of claim 117, wherein the human host is an infant, and the disease is a means of otitis. 121. The method of claim 117 or 120, wherein the human host is an infant, and the disease is meningitis and / or bacteremia. 122. The method of claims 117, 120 or 121, where the human host is an infant, and the disease is pneumonia and / or conjunctivitis. 123. The immunogenetic composition of claims 1 to 113, or a vaccine of claim 115, for use in the treatment or prevention of the disease caused by the infection of Streptococcus pneumoniae. 124. A use of the immunogenetic composition of claims 1 to 113 or a vaccine of claim 115, in the manufacture of a medicament for the treatment or prevention of diseases caused by the infection of Streptococcus pneumoniae. 125. The use of claim 124, wherein the disease is either or both of pneumonia or invasive pneumococcal disease (IPD) of human elderly. 126. The use of claim 124 or 125, wherein the disease are exacerbations of chronic obstructive pulmonary disease (COPD) of human elderly. 127. The use of claim 124, wherein the disease is a means of otitis of human infants. 128. The use of claim 124 or 127, wherein the disease is meningitis and / or bacteremia of human infants. 129. The use of claims 124, 127 or 128, wherein the disease is pneumonia and / or conjunctivitis of human infants. 130. A use of the immunogenetic composition of claims 1 to 113 or a vaccine of claim 115, comprising a capsular saccharide conjugate of the 19F serotype but not comprising the capsular saccharide of serotype 19A in the manufacture of a medicament for the treatment or prevention of diseases caused by Infection of Streptococcus pneumoniae by strains of serotypes 19A. 131. A method for immunizing a human host against the disease caused by infection of the serotype 19A of Streptococcus pneumoniae, comprising the steps of administering to the host an immunoprotective dose of the immunogenic composition of claims 1 to 113 or of the vaccine of the claim 115 which comprises a capsular saccharide conjugate of serotype 19F but does not comprise the capsular saccharide of serotype 19A. 132. The composition of claims 1 to 113, which does not It comprises the capsular saccharide of serotype 1 9F, suitable for the prevention of diseases caused by the infection of Streptococcus pneumoniae by strains of serotype 19F. The vaccine of claim 1, which does not comprise the capsular saccharide of serotype 19A, suitable for the prevention of diseases caused by the infection of Streptococcus pneumoniae by strains of serotype 19A. 1 34. A method for producing a protective immune response in children against the Otitis medium, comprising the administration as separate or combined components, sequentially or concomitantly of (i) an immunogenic composition or a vaccine according to any of claims 1 to 1 1 5 and (i) a protein D of Hemophilus influenzae such protein D can be free and / or conjugated. 135. A method for producing a protective immune response in infants against S. pneumonia by administering the immunogenetic composition or vaccine of any preceding claim. 1 36. A method for producing a protective immune response in the elderly against S. pneumonia by administering, in combination, sequentially or concomitantly (i) the immunogenetic composition or vaccine of any preceding claim, (ii) one or more selected S. pneumoniae surface proteins. of the group consisting of the family (polyhistidine triad) PhtX and pneumolysin. 1 37. A method for producing a protective immune response in infants against the Otitis medium, by administering the composition immunogenetics or vaccine of any previous claim. 138. A method for producing a protective immune response in infants against the Otitis medium by administering, as separate or combined components, sequentially or concomitantly, (i) the vaccine of any preceding claim, (ii) one or more surface proteins of S. pneumoniae. selected from the group consisting of the family family (triad of poly histidine) PhtX and pneumolysin. 139. The immunogenetic composition of claims 1 -1, 3 or the vaccine of claim 1, which comprises the saccharide conjugates derived 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 of 4, 6B, 9V, 14, 18C, 19F and 23F, is not significantly lower than that induced by the Prevnar® vaccine in human receptors 140. The immunogenic composition of claim 1, wherein the GMC antibody titer induced against serotype 4 is not significantly lower than that induced by the Prevnar® vaccine in human receptors 141. The immunogenetic composition of claim 139 or 140, wherein the antibody titer of GMC induced against serotypes 6B is not significantly lower than that induced by the vaccine of Prevnar® in human receptors 142. The immunogenetic composition of claims 1 39-141, wherein the antibody titer of GMC induced against 9V serotypes is not significantly lower than that induced by the vaccine of Prevnar® in human receptors. 143. The immunogenic composition of claims 1 39-142, wherein the antibody titre of G MC induced against serotype 14 is not significantly lower than that induced by the Prevnar® vaccine in human 144. receptors. The immunogenic composition of the claims 139-143, wherein the antibody titer of GMC induced against the serotype 1 8C is not significantly lower than that induced by the vaccine of Prevnar® in human receptors 145. The immunogenetic composition of the claims 1 39-144, wherein the GMC antibody titer induced against the 19F serotype is not significantly lower than that induced by the Prevnar® vaccine in human receptors. 146. The immunogenetic composition of claims 1 39-145, wherein the antibody titer of G MC induced against the 23F serotype is not significantly lower than that induced by the Prevnar® vaccine in human receptors. 147. The immunogenetic composition of claims 1 39-146, which comprises a saccharide conjugate of serotype 3. 148. The immunogenetic composition of the claims 1 39-147, which comprises a saccharide conjugate of serotype 6A. 149. The immunogenetic composition of claims 139-148, which comprises a saccharide conjugate of serotype 1 9A. 50. The ungel genetic composition of claims 1 39-149, which comprises a saccharide conjugate of serotype 22F.