EP2908854A2 - Composition immunogène - Google Patents

Composition immunogène

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Publication number
EP2908854A2
EP2908854A2 EP13776808.1A EP13776808A EP2908854A2 EP 2908854 A2 EP2908854 A2 EP 2908854A2 EP 13776808 A EP13776808 A EP 13776808A EP 2908854 A2 EP2908854 A2 EP 2908854A2
Authority
EP
European Patent Office
Prior art keywords
streptococcus pneumoniae
immunogenic composition
serotype
saccharide
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP13776808.1A
Other languages
German (de)
English (en)
Inventor
Vincent Verlant
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Original Assignee
GlaxoSmithKline Biologicals SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB1218660.7A external-priority patent/GB201218660D0/en
Priority claimed from US13/827,203 external-priority patent/US20140105927A1/en
Priority claimed from US13/826,932 external-priority patent/US20140193451A1/en
Application filed by GlaxoSmithKline Biologicals SA filed Critical GlaxoSmithKline Biologicals SA
Publication of EP2908854A2 publication Critical patent/EP2908854A2/fr
Ceased legal-status Critical Current

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    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
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    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
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    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to improved immunogenic compositions and vaccines, and their use in medicine.
  • the invention relates to immunogenic compositions for use in enhancing antibody-mediated opsonic activity against a Streptococcus pneumoniae serotype or methods for enhancing antibody-mediated opsonic activity against a Streptococcus pneumoniae serotype.
  • Streptococcus pneumoniae also known as the pneumococcus, is a Gram-positive bacterium.
  • S. pneumoniae is a major public health problem all over the world and is responsible for considerable morbidity and mortality, especially among infants, the elderly and immunocompromised persons.
  • S. pneumoniae causes a wide range of important human pathologies including community-acquired pneumonia, acute sinusitis, otitis media, meningitis, bacteremia, septicemia, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, bronchitis and COPD exacerbations and brain abscess.
  • S. pneumoniae is estimated to be the causal agent in 3,000 cases of meningitis, 50, 000 cases of bacteremia, 500,000 cases of pneumonia, and 7,000,000 cases of otitis media annnually in the United States alone
  • IPD Invasive pneumococcal disease
  • pneumoniae is isolated from the blood or another normally sterile site (Musher D M.
  • COPD chronic obstructive pulmonary disease
  • Exacerbations of COPD increase rates of hospitalization and mortality and decrease quality of life. Exacerbations are marked by an increase from baseline in dyspnoea, sputum volume and sputum purulence. Approximately 50% of acute exacerbations of symptoms in COPD are caused by non-typeable Haemophilus influenzae, Moraxella catarrhalis,
  • Streptococcus pneumoniae and Pseudomonas aeruginosa Streptococcus pneumoniae and Pseudomonas aeruginosa.
  • the present invention relates to immunogenic compositions for use in enhancing antibody-mediated opsonic activity against a Streptococcus pneumoniae serotype or methods for enhancing antibody-mediated opsonic activity against a Streptococcus pneumoniae serotype.
  • the inventors have found that Pneumococcal proteins can enhance the antibody- mediated opsonic activity against a serotype of Streptococcus pneumoniae when the pneumococcal protein(s) is co-administered in combination with a Streptococcus pneumoniae capsular saccharide derived from a strain of that serotype.
  • WO02/22167 refers to a results obtained by simultaneously stimulating the cell mediated branch of the immune system and the humoral branch of the immune system. The effect in WO02/22167 would not enhance the antibody-mediated opsonic activity against a strain of Streptococcus pneumoniae, and would not be reflected in enhanced antibody-mediated opsonic activity measured using an
  • OPA opsonophagocytosis
  • opsonophagocytosis OPA assay. This is particularly surprising since Pneumococcal proteins induce little or no antibody mediated opsonic activity when they are injected alone (in the absence of pneumococcal saccharides).
  • an immunogenic composition comprising a Streptococcus pneumoniae protein can enhance the antibody-mediated opsonic activity against a targeted serotype of Streptococcus pneumoniae when the Streptococcus pneumoniae protein is coadministered with a Streptococcus pneumoniae saccharide derived from a strain of the targeted serotype of Streptococcus pneumoniae.
  • immunogenic composition comprising
  • an immunogenic composition comprising
  • an immunogenic composition comprising
  • immunogenic composition comprising
  • Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae.
  • an immunogenic composition comprising (i) at least one Streptococcus pneumoniae protein;
  • the at least one Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of
  • co-administering the (i) at least one Streptococcus pneumoniae protein; and (ii) a Streptococcus pneumoniae capsular saccharide derived from a strain of the targeted serotype of Streptococcus pneumoniae; wherein co-administering the Streptococcus pneumoniae capsular saccharide in combination with the at least one Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae; wherein co-administering is performed by administering an immunogenic composition comprising the Streptococcus pneumoniae capsular saccharide in combination with the at least one Streptococcus pneumoniae protein.
  • a ninth aspect there is provided method of immunising a human host against diseases caused by Streptococcus pneumoniae infection comprising administering to the host an immunoprotective dose of an immunogenic composition comprising (i) at least one
  • Streptococcus pneumoniae protein and (ii) a Streptococcus pneumoniae capsular saccharide derived from a strain of a targeted serotype of Streptococcus pneumoniae;
  • the at least one Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae.
  • Streptococcus pneumoniae capsular saccharide derived from a strain of a targeted serotype of Streptococcus pneumoniae as an immunogenic composition, wherein co-administering the Streptococcus pneumoniae capsular saccharide in combination with the at least one
  • Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae or
  • an increased immune response is measurable as an enhanced antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae.
  • a method of increasing an immune response in a subject against infection or disease caused by Streptococcus pneumoniae comprising:
  • immunogenic composition thereby eliciting an immune response, which immune response is measurable as an enhanced antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae.
  • an immunogenic composition for enhancing antibody-mediated opsonic activity against a targeted serotype of Streptococcus pneumoniae comprising
  • Figure 1 Bar chart showing anti-dPly antibody titres, according to an ELISA assay, in human adults after immunisation with ⁇ g detoxified Pneumolysin (Ply-10), 30 ⁇ g detoxified pneumolysin (Ply-30), ⁇ g of detoxified pneumolysin and ⁇ g of PhtD (PIPh-10), 30 ⁇ g of detoxified pneumolysin and 30 ⁇ g of PhtD (PIPh-3), a vaccine comprising 10 pneumococcal conjugates combined with ⁇ g detoxified pneumolysin and ⁇ g of PhtD (10vPP10), a vaccine comprising 10 pneumococcal conjugates combined with 30 ⁇ g detoxified pneumolysin and 30 ⁇ g of PhtD (10vPP30), and a vaccine comprising 23 unconjugated pneumococcal saccharides.
  • the white bars represent immunogenicity prior to immunisation, the grey bars represent
  • Figure 2 Similar bar chart to Figure 2 but depicting the anti-PhtD titres rather than anti-Ply titres.
  • Figure 3 Bar chart showing anti-PS1 opsonic antibody titres, according to an
  • Pneumolysin Pneumolysin (Ply-10), 30 ⁇ g detoxified pneumolysin (Ply-30), ⁇ g of detoxified pneumolysin and ⁇ g of PhtD (PIPh-10), 30 ⁇ g of detoxified pneumolysin and 30 ⁇ g of PhtD (PIPh-3), a vaccine comprising 10 pneumococcal conjugates combined with ⁇ g detoxified pneumolysin and 10 ⁇ g of PhtD (10vPP10), a vaccine comprising 10 pneumococcal conjugates combined with 30 ⁇ g detoxified pneumolysin and 30 ⁇ g of PhtD (10vPP30), and a vaccine comprising 23 unconjugated pneumococcal saccharides.
  • Figure 5 Similar bar chart to Figure 3 but depicting the anti-PS5 titres rather than the anti-PS1 titers.
  • Figure 6 Similar bar chart to Figure 3 but depicting the anti-PS6B titres rather than the anti- PS 1 titers.
  • Figure 7 Similar bar chart to Figure 3 but depicting the anti-PS7F titres rather than the anti-PS1 titers.
  • Figure 8 Similar bar chart to Figure 3 but depicting the anti-PS9V titres rather than the anti- PS 1 titers.
  • Figure 9 Similar bar chart to Figure 3 but depicting the anti-PS14 titres rather than the anti-PS1 titers.
  • Figure 10 Similar bar chart to Figure 3 but depicting the anti-PS18C titres rather than the anti- PS1 titers.
  • Figure 11 Similar bar chart to Figure 3 but depicting the anti-PS19F titres rather than the anti- PS 1 titers.
  • Figure 12 Similar bar chart to Figure 3 but depicting the anti-PS23F titres rather than the anti- PS 1 titers.
  • Figure 13 Sequence listing Figure 14: Graph comparing the immunogenicity of a composition comprising 12 saccharide conjugates, PhtD, dPIy and PE-PilA (12V + prot) with a composition comprising 12 saccharides conjugates (12V) and a composition comprising 10 saccharide conjugates (10V) as measured after injection of mice using an anti-saccharide ELISA assay.
  • GMC geometric mean concentration .
  • IC confidence intervals.
  • Figure 15 Graph comparing the immunogenicity of a composition comprising 12 saccharide conjugates, PhtD, dPIy and PE-PilA (12V + prot) with a composition comprising 12 saccharides conjugates (12V) and a composition comprising 10 saccharide conjugates (10V) as measured after injection of mice using an opsonophagocytosis assay.
  • GMT geometric means titer.
  • Figure 16 Graph comparing the immunogenicity of a composition comprising 12 saccharide conjugates, PhtD, dPIy and PE-PilA (12V + prot) with a composition comprising PhtD, dPIy and PE-PilA alone (prot) as measured after injection of mice using an anti-protein ELISA assay.
  • GMC geometric mean concentration .
  • IC confidence intervals.
  • Figure 17 Graph comparing the immunogenicity of a composition comprising 12 saccharide conjugates, PhtD, dPIy and PE-PilA (12V + prot) with a composition comprising 12 saccharides conjugates (12V) and a composition comprising 10 saccharide conjugates (10V) as measured after injection of guinea pigs using an opsonophagocytosis assay.
  • GMT geometric means titer.
  • Figure 18 Graph comparing the immunogenic of a composition comprising 12 saccharide conjugates, PhtD, dPIy and PE-PilA (12V + prot) with a composition comprising 12 saccharides conjugates (12V) and a composition comprising 10 saccharide conjugates (10V) as measured after injection of guinea pigs using an anti-saccharide ELISA.
  • GMC geometric mean concentration .
  • IC confidence intervals.
  • Figure 19 Graph comparing the immunogenic of a composition comprising 12 saccharide conjugates, PhtD, dPIy and PE-PilA (12V + prot) with a composition comprising PhtD, dPIy and PE-PilA alone (prot) as measured after injection of guinea pigs using an anti-protein ELISA.
  • Opsonic antibodies have been shown to be a critical protection mechanism induced by saccharide-based pneumococcal vaccines.
  • Opsonophagocytosis assays have been developed and validated to characterize and assess current and possible future pneumococcal vaccines (Validation of a routine opsonophagocytosis assay to predict invasive pneumococcal disease efficacy of conjugate vaccine in children; Henckaerts et al; Vaccine 25 (2007):2518-2527).
  • opsonophagocytosis assays involve incubating serum obtained from a subject together with opsonophagocytic cells, a source of complement, and bacteria of the targeted Streptococcus pneumoniae serotype.
  • the components of the sera promote opsonophagocytosis of the bacteria and the extent to which these components promote opsonophagocytosis can be measured by determining the number of remaining (not 'killed') bacteria at the end of the incubation. This is a measure of the opsonic activity of the sera and thus the antibodies raised.
  • Antibodies which can bind to the bacteria promote opsonophagocytic 'killing' of the bacteria, for example, by binding of the Fc regions of the antibodies to Fc receptors on the surface of phagocytic cells enabling the phagocytic cell to engulf bacteria coated with antibodies.
  • the present inventors observed immunizing human subjects with a vaccine comprising pneumococcal saccharides and a higher dose of pneumococcal proteins trends towards a higher opsonic activity against the serotypes from which the saccharides are derived compared to immunisation of subjects with a vaccine comprising pneumococcal saccharides and a lower dose of pneumococcal proteins. This cannot be due to the presence of humoral immunity since the in vitro OPA assay occurs in the absence of the humoral immune system.
  • the terms 'higher dose' and 'lower dose' can be considered to be relative terms, in the examples of the present application the inventors have demonstrated that the antibody- mediated opsonic activity of a composition comprising 10 saccharides is higher when the saccharides are administered with pneumococcal proteins at doses of 30 ⁇ g than when the saccharides are administered with pneumococcal proteins at dose of ⁇ g, however a similar effect could be seen if the saccharides were administered with different doses of the proteins, what is important is that increasing the dose of pneumococcal proteins leads to higher antibody -mediated opsonic activity.
  • the term 'higher dose' can be considered to refer to a dose of 30 ⁇ g and the term 'lower dose' can be considered to refer to a dose of ⁇ g.
  • this effect could be explained, for example, by the pneumococcal protein having some kind of 'adjuvant' effect on the pneumococcal saccharides, for example the pneumococcal protein may have a stabilising effect on a key epitope found within the saccharides.
  • pneumococcal proteins elicit a small level of opsonic activity that is not normally significant (and thus has not been previously recognized), but when the proteins are administered in the presence of pneumococcal saccharides the opsonic activity of the proteins is added to the opsonic activity to the saccharides leading to a statistically significant opsonic activity and greater immunity.
  • an immunogenic composition comprising
  • the term 'targeted serotype' refers to the serotype from which the Streptococcus pneumoniae capsular saccharide is derived optionally this includes serotypes which are cross- reactive with the serotype from which the Streptococcus pneumoniae capsular saccharide is derived.
  • the targeted serotype is a serotype 19F Streptococcus pneumoniae strain or may be a serotype 19A Streptococcus pneumoniae strain (as some serotype 19A capsular saccharide conjugates can provide protection against 19F disease, i.e. 19F saccharide conjugates are cross-reactive).
  • Streptococcus pneumoniae' refers to a polysaccharide or oligosaccharide which can be isolated from the capsule of the targeted serotype of Streptococcus pneumoniae.
  • Saccharide structures have been isolated from serotypes of Streptococcus pneumoniae:
  • the term 'antibody mediated opsonic activity' refers to opsonic activity as measured using an in vitro opsonophagocytosis (OPA) assay (as described above).
  • OPA in vitro opsonophagocytosis
  • 'enhancing antibody-mediated opsonic activity' means that the immunogenic composition of the invention raises the antibody-mediated opsonic activity.
  • An enhancement in the antibody mediated opsonic activity against the targeted strain can be measured by immunizing a subject such as a human with a Streptococcus pneumoniae saccharide and a Streptococcus pneumoniae protein, taking a sample of the serum from the subject, measuring the opsonic activity with an OPA assay using bacteria from the targeted serotype and comparing this with the opsonic activity of a reference serum sample.
  • a reference serum sample is a sample of serum obtained by immunizing a subject with an equivalent dose of the Streptococcus pneumoniae saccharide either alone, or in the presence of a lower dose of the at least one Streptococcus pneumoniae protein. If the opsonic activity measured is higher for the sample serum than for the reference serum the antibody mediated opsonic activity against the targeted strain is enhanced.
  • the antibody mediated opsonic activity against a targeted serotype can be measured using an opsonophagocytosis assay such as that described in example 2.
  • the antibody-mediated opsonic activity against a targeted serotype after immunisation using an immunogenic composition of the invention is greater than 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 or 2.5 times higher than the antibody-mediated opsonic activity against the targeted serotype of the reference sample.
  • the term 'equivalent dose' means a dose which is the same or within 10% of the reference dose.
  • the antibody-mediated opsonic activity against a targeted serotype is the same when the protein is present and the protein is absent, and yet the level of antibodies raised is higher when the Streptococcus pneumoniae saccharide is administered in combination with a higher dose of the Streptococcus pneumoniae protein compared with the level of antibodies raised when the Streptococcus pneumoniae saccharide is administered in combination with a lower dose of the Streptococcus pneumoniae protein.
  • an immunogenic composition comprising
  • a Streptococcus pneumoniae capsular saccharide derived from a strain of a targeted serotype of Streptococcus pneumoniae for use in immunising a subject so as to enhance antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae.
  • an immunogenic composition comprising (i) at least one Streptococcus pneumoniae protein;
  • an immunogenic composition comprising
  • an immunogenic composition comprising
  • an immunogenic composition comprising (i) at least one Streptococcus pneumoniae protein;
  • an immunogenic composition comprising (i) at least one Streptococcus pneumoniae protein;
  • the at least one Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of
  • a ninth aspect there is provided a method of immunising a human host against diseases caused by Streptococcus pneumoniae infection comprising administering to the host an immunoprotective dose of an immunogenic composition comprising (i) at least one
  • Streptococcus pneumoniae protein and (ii) a Streptococcus pneumoniae capsular saccharide derived from a strain of a targeted serotype of Streptococcus pneumoniae;
  • the at least one Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae.
  • a method of increasing an immune response in a subject against infection or disease caused by Streptococcus pneumoniae comprising: co-administering (i) at least one Streptococcus pneumoniae protein; and (ii) a
  • Streptococcus pneumoniae capsular saccharide derived from a strain of a targeted serotype of Streptococcus pneumoniae as an immunogenic composition, wherein co-administering the Streptococcus pneumoniae capsular saccharide in combination with the at least one
  • Streptococcus pneumoniae protein enhances antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae or
  • an increased immune response is measurable as an enhanced antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae.
  • an immunogenic composition for enhancing antibody-mediated opsonic activity against a targeted serotype of Streptococcus pneumoniae comprising
  • the term 'co-administering' or 'co-administration' refers to administering a dose of two or more components on the same day, this may be carried out by combining the components into a single vaccine, for example in a single syringe, alternatively this could be achieved by administering one or more components in a first composition and one or more components in a second composition on the same day (for example in different limbs of a human subject).
  • the term 'immunogenic composition' may refer to a kit in which one or more components are present in a first container (such as a vial or syringe) and one or more components are present in a second container, for example the kit may contain a composition comprising the pneumococcal saccharides in a first container and the pneumococcal proteins in a second container.
  • a first container such as a vial or syringe
  • the kit may contain a composition comprising the pneumococcal saccharides in a first container and the pneumococcal proteins in a second container.
  • the antibody-mediated opsonic activity against the targeted strain of Streptococcus pneumoniae is enhanced if the antibody-mediated opsonic activity against the targeted serotype of
  • Streptococcus pneumoniae is greater when the Streptococcus pneumoniae capsular saccharide is co-administered with the Streptococcus pneumoniae protein than antibody-mediated opsonic activity elicited by administering an equivalent dose of the Streptococcus pneumoniae capsular saccharide alone (in the absence of the at least one Streptococcus pneumoniae protein).
  • the antibody-mediated opsonic activity against the targeted serotype of Streptococcus pneumoniae is enhanced if the antibody-mediated opsonic activity against the targeted strain of Streptococcus pneumoniae is greater than the antibody-mediated opsonic activity elicited by administering an equivalent dose of the Streptococcus pneumoniae capsular saccharide together with a lower dose of the at least one Streptococcus pneumoniae protein.
  • the at least one Streptococcus pneumoniae protein comprises detoxified pneumolysin (dPIy).
  • pneumolysin or “Ply”, it is meant: native or wild-type pneumolysin from
  • pneumolysin is native or wild-type pneumolysin from pneumococcus or recombinant pneumolysin.
  • Ply is a 53 kDa pore-forming toxin and as such has lytic effects on many mammalian cell types, and at sublytic concentrations Ply has numerous other effects, including complement activation in the absence of antipneumolysin antibodies and induction of pro-inflammatory mediators (Hirst et al., 2000; Mitchell & Andrew, 1997; Paton et al., 1984; Zysk et al., 2001 ).
  • Complement activation is independent of the haemolytic activity of the toxin (Berry et al., 1995).
  • Pneumolysin is now known to interact directly with Toll-like receptor 4 (TLR-4) and known to signal via myeloid differentiation marker 88 to induce production of tumour necrosis factor alpha and interleukin 6 (Malley et al., 2003; Trzcinski et al., 2008).
  • TLR-4 Toll-like receptor 4
  • Myeloid differentiation marker 88 myeloid differentiation marker 88 to induce production of tumour necrosis factor alpha and interleukin 6 (Malley et al., 2003; Trzcinski et al., 2008).
  • Pneumolysin is produced by all known clinical isolates of S. pneumoniae regardless of serotype and
  • pneumolysin is Seq ID No. 34 of WO2010/071986.
  • pneumolysin is Seq ID No. 35 of WO2010/071986.
  • pneumolysin is Seq ID No. 36 of
  • pneumolysin is Seq ID No. 37 of WO2010/071986. In another aspect, pneumolysin is Seq ID No. 41 of WO2010/071986.
  • EP1601689B1 describes methods for purifying bacterial cytolysins such as pneumococcal pneumolysin by chromatography in the presence of detergent and high salt.
  • the term 'pneumolysin' includes fragments and/or variants of pneumolysin, having differences in nucleic acid or amino acid sequences as compared to a wild type sequence.
  • fragments of pneumolysin are used, these fragments will be at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues in length.
  • immunogenic fragments of pneumolysin comprise at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues of the full length sequence, wherein said polypeptide is capable of eliciting an immune response specific for said amino acid sequence.
  • Pneumolysin is known to consist of four major structural domains (Rossjohn et al. Cell. 1997 May 30; 89(5):685-92). These domains may be modified by removing and/or modifying one or more of these domains.
  • a variant of pneumolysin includes sequences in which one or more amino acids are substituted and/or deleted and/or inserted compared to the wild type sequence. Amino acid substitution may be conservative or non-conservative. In one aspect, amino acid substitution is conservative. Substitutions, deletions, insertions or any combination thereof may be combined in a single variant so long as the variant is an
  • variants of pneumolysin typically include any pneumolysin or any fragment of pneumolysin which shares at least 80, 90, 94, 95, 98, or 99% amino acid sequence identity with a wild-type pneumolysin sequence, e.g. SEQ IDs 2-42 from WO2010/071986 (for example SEQ IDs 34, 35, 36, 37, 41 ).
  • variants of pneumolysin typically include any pneumolysin or any fragment of pneumolysin which shares at least 80, 90, 94, 95, 98, or 99% amino acid sequence identity with SEQ ID 36 from WO2010/07198.
  • the present invention includes fragments and/or variants in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2, 1 , up to 10, up to 20, up to 30 or up to 50 amino acids are substituted, deleted, or added in any combination.
  • the present invention includes fragments and/or variants which comprise a B-cell or T-cell epitope.
  • Such epitopes may be predicted using a combination of 2D-structure prediction, e.g. using the PSIPRED program (from David Jones, Brunei Bioinformatics Group, Dept. Biological Sciences, Brunei University, Uxbridge UB8 3PH, UK) and antigenic index calculated on the basis of the method described by Jameson and Wolf (CABIOS 4:181-186 [1988]).
  • immunogenic composition of the invention comprises a variant of pneumolysin, for example, those described in WO05/108580, WO05/076696, W010/071986.
  • fragment as used in this specification is a moiety that is capable of eliciting a humoral and/or cellular immune response in a host animal. Fragments of a protein can be produced using techniques known in the art, e.g. recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Typically, fragments comprise at least 10, 20, 30, 40 or 50 contiguous amino acids of the full length sequence.
  • Fragments may be readily modified by adding or removing 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 amino acids from either or both of the N and C termini.
  • conservative amino acid substitution involves substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and without resulting in decreased immunogenicity.
  • these may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • amino acid modifications to the sequence of a polypeptide may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide.
  • the term “deletion” as used in this specification is the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 1 to 6 residues (e.g. 1 to 4 residues) are deleted at any one site within the protein molecule.
  • the term “insertion” as used in this specification is the addition of one or more non- native amino acid residues in the protein sequence. Typically, no more than about from 1 to 6 residues (e.g. 1 to 4 residues) are inserted at any one site within the protein molecule.
  • pneumolysin and its fragments and/or variants thereof have an amino acid sequence sharing at least 80, 85, 90, 95, 98, 99 or 100% identity with the wild type sequence for pneumolysin, e.g. SEQ IDs 34, 35, 36, 37, 41 from
  • pneumolysin and its fragments and/or variants thereof comprise at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues of the wild type sequence for pneumolysin.
  • Pneumolysin is administered after being detoxified (i.e. rendered non-toxic to a human when provided at a dosage suitable for protection).
  • detoxified i.e. rendered non-toxic to a human when provided at a dosage suitable for protection.
  • dPIy refers to detoxified pneumolysin suitable for medical use (i.e. non toxic).
  • Pneumolysin may be detoxified chemically and/or genetically.
  • Detoxification of pneumolysin can be conducted by chemical means, e.g. using a crosslinking agent, such as formaldehyde, glutaraldehyde and a cross-linking reagent containing an N-hydroxysuccinomido ester and/or a maleimide group (e.g. GMBS) or a combination of these.
  • a crosslinking agent such as formaldehyde, glutaraldehyde and a cross-linking reagent containing an N-hydroxysuccinomido ester and/or a maleimide group (e.g. GMBS) or a combination of these.
  • the pneumolysin is detoxified by exposure to formaldehyde.
  • Such methods are well known in the art for various toxins; see for example EP1601689B1 , WO04/081515, WO2006/032499.
  • the pneumolysin used in chemical detoxification may be a native or recombinant protein or a protein that has been genetically engineered to reduce its toxicity (see below). Fusion proteins of pneumolysin or fragments and/or variants of pneumolysin may also be detoxified by chemical means. Therefore, in an embodiment, immunogenic compositions of the invention may comprise pneumolysin which has been chemically detoxified, e.g. by a formaldehyde treatment.
  • Pneumolysin can also be genetically detoxified.
  • the invention encompasses pneumococcal proteins which may be, for example, mutated proteins.
  • mutated is used herein to mean a molecule which has undergone deletion, addition or substitution of one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids), for example by using well known techniques for site directed mutagenesis or any other conventional method.
  • the molecule has undergone deletion or substitution of 1-15, suitably 10-15 amino acids.
  • the mutated sequences may remove undesirable activities such as membrane permeation, cell lysis, and cytolytic activity against human erythrocytes and other cells, in order to reduce the toxicity, whilst retaining the ability to induce anti-pneumolysin protective and/or neutralizing antibodies following administration to a human.
  • Fusion proteins of pneumolysin or fragments and/or variants of pneumolysin may also be detoxified by genetic means. Any of these modifications may be introduced using standard molecular biology and biochemical techniques. For example, as described above, a mutant pneumolysin protein may be altered so that it is biologically inactive whilst still maintaining its immunogenic epitopes, see, for example, WO90/06951 , Berry et al.
  • a pneumolysin protein may be detoxified by three amino acid substitutions comprising T 65 to C, G 293 to C and C 248 to A.
  • Another example of a genetically detoxified pneumolysin that can be used in the present invention is SEQ ID 9 from WO201 1/075823.
  • immunogenic compositions of the invention may comprise pneumolysin which has been genetically detoxified.
  • immunogenic compositions of the invention may comprise pneumolysin which has been chemically and genetically detoxified.
  • the immunogenic composition comprises 26 ⁇ g-45 ⁇ g (for example 26 ⁇ g-40 ⁇ g, 27 ⁇ g-39 ⁇ g, 27 ⁇ g-37 ⁇ g, 28 ⁇ g-35 ⁇ g, 29 ⁇ g-33 ⁇ g or around 30 ⁇ g) of pneumolysin, per human dose.
  • the at least one Streptococcus pneumoniae protein comprises PhtD (Poly Histidine Triad protein D).
  • PhtD Poly Histidine Triad protein D
  • the terms 'Pneumococcal Histidine Triad' and 'Poly Histidine Triad' have both been used to describe the PhtX family which includes PhtD. For this reason the terms 'Pneumococcal Histidine Triad' and 'Poly Histidine Triad' can be considered to be equivalent to one another and therefore interchangeable for the purposes of the present invention.
  • the term "PhtD" as used herein includes the full length protein with the signal sequence attached or the mature full length protein with the signal peptide (for example 20 amino acids at
  • PhtD is also referred to "Sp036D".
  • PhtD is the full length protein with the signal sequence attached e.g. SEQ ID NO: 4 of WOOO/37105.
  • PhtD is a sequence comprising the mature full length protein with the signal peptide (for example 20 amino acids at N-terminus) removed, e.g. amino acids 21-838 of SEQ ID NO: 4 of WOOO/37105 (or SEQ ID NO: 1 of the present application).
  • the PhtD sequence comprises an N- terminal methionine.
  • the present invention also includes PhtD polypeptides which are immunogenic fragments of PhtD, variants of PhtD and/or fusion proteins of PhtD.
  • PhtD polypeptides which are immunogenic fragments of PhtD, variants of PhtD and/or fusion proteins of PhtD.
  • these fragments will be at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues in length, e.g from a PhtD amino acid sequence in
  • immunogenic fragments of PhtD protein comprise at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues of the sequence shown in SEQ ID NO: 4 of WOOO/37105, wherein said polypeptide is capable of eliciting an immune response specific for said amino acid sequence.
  • the immunogenic composition of the invention comprises a fragment of PhtD, for example described in
  • each fragment optionally contains one or more histidine triad motif(s) of such polypeptides.
  • a histidine triad motif is the portion of polypeptide that has the sequence HxxHxH (SEQ ID NO:2) where H is histidine and x is an amino acid other than histidine.
  • the or each fragment contains exactly or at least 2, 3, 4 or 5 histidine triad motifs (optionally, with native PhtD sequence between the 2 or more triads, or intra-triad sequence) where the fragment is more than 50, 60, 70, 80, 85, 90, 95, 98, 99 or 100% identical to a native pneumococcal intra-triad PhtD sequence (e.g. the intra- triad sequence shown in SEQ ID NO: 4 of WOOO/37105).
  • Fragments of PhtD proteins optionally contain one or more coiled coil regions of such polypeptides.
  • a coiled coil region is a region predicted by "Coils" algorithm Lupus, A et al (1991 ) Science 252; 1 162-1 164.
  • the or each fragment contains exactly or at least 2, 3 or 4 coiled coil regions.
  • the or each fragment contains exactly or at least 2, 3 or 4 coiled coil regions where the fragment is more than 50, 60, 70, 80, 90, 95, 96, 98, 99 or 100% identical to a native pneumococcal PhtD sequence (e.g. the sequence shown in SEQ ID NO: 4 of WOOO/37105).
  • the or each fragment includes one or more histidine triad motif as well as at least 1 , 2, 3 or 4 coiled coil regions.
  • the variation is generally in a portion thereof other than the histidine triad residues and the coiled-coil region, although variations in one or more of these regions may be made.
  • a polypeptide variant includes sequences in which one or more amino acids are substituted and/or deleted and/or inserted compared to the wild type sequence. Amino acid substitution may be conservative or non-conservative. In one aspect, amino acid substitution is conservative.
  • variants of PhtD typically include any fragment or variation of PhtD which shares at least 80, 90, 95, 96, 98, or 99% amino acid sequence identity with a wild-type PhtD sequence, e.g. SEQ ID NO: 4 of WO00/37105.
  • the present invention includes fragments and/or variants in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2,1 , up to 10, up to 20, up to 30, or up to 50 amino acids are substituted, deleted, or added in any combination.
  • the present invention includes fragments and/or variants which comprise a B-cell or T-cell epitope. Such epitopes may be predicted using a combination of 2D-structure prediction, e.g. using the
  • Variants can be produced by conventional molecular biology techniques. Variants as used herein may also include naturally occurring PhtD alleles from alternate Streptococcus strains that exhibit polymorphisms at one or more sites within the homologous PhtD gene.
  • PhtD and its fragments, variants and/or fusion proteins thereof comprise an amino acid sequence sharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity with amino acid sequence 21 to 838 of SEQ ID NO:4 of WO00/37105 (SEQ ID NO: 1 ).
  • PhtD and its fragments, variants and/or fusion proteins thereof have an amino acid sequence sharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity with amino acid sequence 21 to 838 of SEQ ID NO:4 of WO00/37105 (SEQ ID NO: 1 ).
  • PhtD and its fragments, variants and/or fusion proteins thereof comprise an amino acid sequence having an N-terminal methionine.
  • PhtD and its fragments, variants and/or fusion proteins thereof comprise at least about 15, at least about 20, at least about 40, or at least about 60 or at least about 100, or at least about 200, or at least about 400 or at least about 800 contiguous amino acid residues of the sequence shown in SEQ ID NO: 4 of WO00/37105.
  • PhtD and its fragments, variants and/or fusion proteins thereof comprise an amino acid sequence sharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity with amino acid sequence SEQ ID NO:73 of WO00/39299.
  • PhtD and its fragments, variants and/or fusion proteins thereof have an amino acid sequence sharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity with amino acid sequence SEQ ID NO:73 of WO00/39299.
  • PhtD and its fragments, variants and/or fusion proteins thereof comprise at least about 15, at least about 20, at least about 40, or at least about 60, or at least about 100, or at least about 200, or at least about 400 or at least about 800 contiguous amino acid residues of the sequence shown in SEQ ID NO: 73 of WO00/39299.
  • the PhtD sequence is SEQ ID NO. 1 or 5 from WO2011/075823.
  • the present invention also includes PhtD proteins which differ from naturally occurring S. pneumoniae polypeptides in ways that do not involve the amino acid sequence.
  • Non- sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.
  • those with modifications which increase peptide stability such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence.
  • analogs that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring or synthetic amino acids, e.g. ⁇ or ⁇ amino acids, and cyclic analogs.
  • the immunogenic composition comprises 26 ⁇ g-45 ⁇ g (for example 26 ⁇ g-40 ⁇ g, 27 ⁇ g-39 ⁇ g, 27 ⁇ g-37 ⁇ g, 28 ⁇ g-35 ⁇ g, 29 ⁇ g-33 ⁇ g or around 30 ⁇ g) of PhtD, per human dose.
  • the uses, methods and immunogenic compositions of the invention provide a an enhanced antibody mediated immune response, for these reasons the uses, methods and immunogenic compositions of the invention are likely to be beneficial, in particular, to subjects or human hosts which are immunocompromised, as the enhancement effect may increase the likelihood that immunocompromised subjects or human hosts are able to respond to the vaccine whilst they may not respond to other vaccines. Similarly this may be true of individuals who are poor responders to vaccination with polysaccharide (as the enhancement effect boost the response to the saccharide).
  • the subject or human host may be an immunocompromised human.
  • the term 'immunocompromised' refers to a human who is incapable of developing or unlikely to develop a robust immune response, usually as a result of disease, malnutrition or
  • immunosuppressive therapy Those who can be considered to be immunocompromised include, but are not limited to, subjects with AIDS (or HIV positive), subjects with severe combined immune deficiency (SCID), diabetics, subjects who have had transplants and who are taking immunosuppressive drugs, and those that are receiving chemotherapy. Immunocompromised individuals also includes subjects with most forms of cancer (other than skin cancer), sickle cell anemia, cystic fibrosies, those who do not have a spleen, subjects with endstage kidney disease (dialysis), and those who have been taking corticosteroids on a frequent basis by pill or injection within the last year. Subjects with severe liver, lung or heart disease also may be immunocompromised.
  • the immunocompromised human is a human who has been diagnosed with HIV infection, chronic renal failure, nephritic syndrome, a disease associated with treatment with immunosuppressive drugs or radiation therapy, or congenital immunodeficiency.
  • the disease associated with treatment with immunosuppressive drugs or ration therapy is selected from the group consisting of malignant neoplasms, leukemias, lymphomas, and Hodgkin disease.
  • the subject or human host is a human that has been characterized as being an immunocompromised human.
  • the term 'has been characterized' means that the subject or human host has been determined to be immunocompromised, for example by a diagnosis of an immunocompromised condition by a doctor or other medical practitioner.
  • the methods of the invention comprise a step of characterizing the human host or subject as an immunocompromised human.
  • the subject or human host may be a poor responder to immunisation with polysaccharide.
  • the subject or human host is a human that has been characterized as being a poor responder to immunisation with polysaccharide.
  • the subject or human host is an elderly human (optionally wherein the elderly human is more than 50, 55, 60, 65, 70, 75 or 80 years old).
  • the host or subject is a human adult, an adult human is a human greater than 18 years of age.
  • the host or subject is an elderly human suitably an elderly person aged 60, 62, 65, 67, or 70 years and over.
  • the host or subject is a younger adult (i.e.
  • the host or subject is an infant human such as a child more than 6 months old, especially children 6 to 23 months of age.
  • the host or subject is a toddler such as a child between 24 months and 3 years of age
  • human dose means a dose which is in a volume suitable for human use.
  • the final dose volume (vaccine composition volume) administered to a human patient may be between 0.25 to 1.5 ml, 0.2 to 1.0 ml, or 0.4 to 0.6 ml.
  • a human dose is 0.5 ml.
  • a human dose is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml.
  • a human dose is between 1 ml and 1.5 ml.
  • a human dose may be less than 0.5 ml such as between 0.25 and 0.5 ml.
  • the immunogenic compositions of the invention may comprise at least one further unconjugated or conjugated Streptococcus pneumoniae protein.
  • S. pneumoniae and Streptococcus pneumoniae are considered to be interchangeable and equivalent to one another.
  • the at least one further unconjugated or conjugated Streptococcus pneumoniae protein is selected from the group consisting of Poly Histidine Triad family (PhtX), Choline Binding Protein Family (CbpX), CbpX truncates, LytX (autolysin encoding gene) family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins, PcpA (pneumococcal choline binding protein A), PspA (pneumococcal surface protein A), PsaA (pneumococcal surface adhesion protein A) , Sp128 (streptococcus pneumoniae 128), Sp101 (streptococcus pneumoniae 101 ), Sp130(streptococcus pneumoniae 130), SP125 (streptococcus pneumoniae 125) and SP133(streptococcus pneumoniae 133).
  • PcpA pneu
  • the at least one further unconjugated or conjugated Streptococcus pneumoniae protein is selected from the group consisting of Choline Binding Protein Family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins, PcpA, PspA, PsaA, Sp128, Sp101 , Sp130, SP125 and SP133.
  • the Pht Poly Histidine Triad also known as Poly Histidine Triad
  • family comprises proteins PhtA (Poly Histidine Triad family A), PhtB (Poly Histidine Triad family B), PhtD (Poly Histidine Triad family B), and
  • PhtE Poly Histidine Triad family E
  • the family is characterized by a lipidation sequence, two domains separated by a proline-rich region and several histidine triads, possibly involved in metal or nucleoside binding or enzymatic activity, (3-5) coiled-coil regions, a conserved N- terminus and a heterogeneous C terminus. It is present in all strains of pneumococci tested. Homologous proteins have also been found in other Streptococci and Neisseria.
  • the Pht protein of the invention is PhtD.
  • phrases Pht A, B, D, and E refer to proteins having sequences disclosed in the citations below as well as naturally-occurring (and man-made) variants thereof that have a sequence homology that is at least 90% identical to the referenced proteins. Optionally it is at least 95% identical or at least 97% identical.
  • PhtA is disclosed in WO 98/18930, and is also referred to Sp36. As noted above, it is a protein from the Poly Histidine triad family and has the type II signal motif of LXXC (SEQ ID NO:3).
  • PhtD is disclosed in WO 00/37105, and is also referred to Sp036D. As noted above, it also is a protein from the Poly Histidine triad family and has the type II LXXC signal motif.
  • PhtB is disclosed in WO 00/37105, and is also referred to Sp036B. Another member of the PhtB family is the C3-Degrading Polypeptide, as disclosed in WO 00/17370.
  • This protein also is from the Poly Histidine triad family and has the type II LXXC signal motif.
  • an immunologically functional equivalent is the protein Sp42 disclosed in WO 98/18930.
  • a PhtB truncate (approximately 79kD) is disclosed in W099/15675 which is also considered a member of the PhtX family.
  • PhtE is disclosed in WOOO/30299 and is referred to as BVH-3.
  • any Pht protein is referred to herein, it is meant that immunogenic fragments or fusions thereof of the Pht protein can be used.
  • a reference to PhtX includes immunogenic fragments or fusions thereof from any Pht protein.
  • a reference to PhtD or PhtB is also a reference to PhtDE or PhtBE fusions as found, for example, in WO0198334.
  • phosphorylcholine moieties of cell wall teichoic acid and membrane-associated lipoteichoic acid Structurally, they have several regions in common over the entire family, although the exact nature of the proteins (amino acid sequence, length, etc.) can vary.
  • choline binding proteins comprise an N terminal region (N), conserved repeat regions (R1 and/or R2), a proline rich region (P) and a conserved choline binding region (C), made up of multiple repeats, that comprises approximately one half of the protein.
  • CbpX Choline Binding Protein family
  • PbcA is disclosed in W098/21337.
  • SpsA is a Choline binding protein disclosed in WO 98/39450.
  • the Choline Binding Proteins are selected from the group consisting of CbpA, PbcA, SpsA and PspC.
  • An embodiment of the invention comprises CbpX truncates wherein "CbpX” is defined above and “truncates” refers to CbpX proteins lacking 50% or more of the Choline binding region (C).
  • CbpX CbpX proteins lacking 50% or more of the Choline binding region (C).
  • such proteins lack the entire choline binding region.
  • the such protein truncates lack (i) the choline binding region and (ii) a portion of the N-terminal half of the protein as well, yet retain at least one repeat region (R1 or R2).
  • the truncate has 2 repeat regions (R1 and R2).
  • NR1xR2 and R1xR2 as illustrated in W099/51266 or W099/51 188, however, other choline binding proteins lacking a similar choline binding region are also contemplated within the scope of this invention.
  • the LytX family is membrane associated proteins associated with cell lysis.
  • the N- terminal domain comprises choline binding domain(s), however the LytX family does not have all the features found in the CbpA family noted above and thus for the present invention, the LytX family is considered distinct from the CbpX family.
  • the C- terminal domain contains the catalytic domain of the LytX protein family.
  • the family comprises LytA, B and C.
  • LytA is disclosed in Ronda et al., Eur J Biochem, 164:621-624 (1987).
  • LytB is disclosed in WO 98/18930, and is also referred to as Sp46.
  • LytC is also disclosed in WO 98/18930, and is also referred to as Sp91.
  • An embodiment of the invention comprises LytC.
  • LytX truncates wherein "LytX” is defined above and “truncates” refers to LytX proteins lacking 50% or more of the Choline binding region. Optionally such proteins lack the entire choline binding region.
  • CbpX is selected from the group consisting of CbpA, PbcA, SpsA and PspC.
  • it is CbpA.
  • LytX is LytC (also referred to as Sp91 ).
  • Another embodiment of the present invention is a PspA or PsaA truncate lacking the choline binding domain (C) and expressed as a fusion protein with LytX.
  • LytX is LytC.
  • PsaA and PspA both are know in the art.
  • PsaA and transmembrane deletion variants thereof have been described by Berry & Paton, Infect Immun 1996 Dec;64(12):5255-62.
  • PspA and transmembrane deletion variants thereof have been disclosed in, for example, US 5804193, WO 92/14488, and WO 99/53940.
  • PcpA this protein is known in the art, for example PcpA has been described in WO201 1/075823.
  • the term 'PcpA refers to a protein comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:2 or 7 from WO2011/075823 or fragments of at least 100, 150, 200, 25 or more consecutive amino acids of SEQ ID NO:2 or 7 from WO201 1/075823.
  • Sp128 and Sp130 are disclosed in WO00/76540.
  • Sp125 is an example of a
  • S. pneumoniae saccharide (described in the section entitled S. pneumoniae saccharide conjugates below).
  • one or more S.pneumoniae proteins is conjugated to a saccharide from a different bacterium.
  • the term 'conjugated to' in this context means that the protein is covalently bonded to a saccharide, in this situation the protein is acting as a carrier protein.
  • the at least one further unconjugated or conjugated S.pneumoniae protein comprises a Poly Histidine family (PhtX) protein selected from the group consisting of PhtB, PhtE, PhtA, or a PhtBD or PhtDE fusion protein.
  • the composition comprises 1 ⁇ 9-100 ⁇ 9, 1 ⁇ 9-75 ⁇ 9, 1 ⁇ 9-50 ⁇ 9, ⁇ g- 25 ⁇ 9, 1 ⁇ 9-20 ⁇ 9, 5 ⁇ 9-15 ⁇ 9, 25 ⁇ 9-40 ⁇ 9, 28 ⁇ 9-35 ⁇ 9, around 10 ⁇ 9 or around 30 ⁇ 9 of the at least one further unconjugated or conjugated S. pneumoniae protein, per human dose, per protein.
  • the Streptococcus pneumoniae capsular saccharide is a capsular saccharide from a serotype selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 15, 16B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • a serotype selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 15, 16B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • the Streptococcus pneumoniae capsular saccharide is a capsular saccharide from a serotype selected from the group consisting of 4, 6B, 7F, 9V, 18C, 19F, and 23F.
  • the immunogenic composition of the invention may further comprise 1 or more (e.g. 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23) further Streptococcus pneumoniae capsular saccharide conjugates.
  • the 1 or more further Streptococcus pneumoniae capsular saccharide conjugates may be selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 15, 16B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • the term capsular saccharide includes capsular polysaccharides and oligosaccharides derivable from the capsular polysaccharide. An oligosaccharide contains at least 4 sugar residues.
  • conjugate and conjugated relate to a capsular saccharide covalently bonded to a carrier protein.
  • the total number of saccharide serotypes is less than or equal to 23, optionally the immunogenic composition comprising 10-23 serotypes, 10-16, serotypes 10-15 serotypes, 10-14 serotypes, 10-13 serotypes or 10-12 serotypes.
  • the immunogenic composition comprises less than 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14 or 13 Streptococcus pneumoniae capsular saccharide conjugates.
  • the immunogenic composition further comprises unconjugated Streptococcus pneumoniae saccharides such that the number of conjugated and unconjugated saccharide serotypes is less than or equal to 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, or 1 1.
  • the multivalent immunogenic composition of the invention comprises saccharides from the following serotypes 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, although it is appreciated that one or two other serotypes could be substituted depending on the age of the recipient receiving the immunogenic composition and the geographical location where the immunogenic composition will be administered.
  • a 7 valent immunogenic composition may comprise
  • a 10 valent immunogenic composition may comprise the same 7 serotypes and further comprise saccharides derived from serotypes 1 , 5 and 7F.
  • a 12 valent immunogenic composition may comprise the same 10 serotypes and further comprises saccharides derived from serotypes 6A and 19A.
  • a 13 valent immunogenic composition may comprise the same 12 serotypes and further comprise a serotype 3 saccharide.
  • a 15 valent immunogenic composition may comprise the same 13 serotypes and further comprise saccharides derived from serotypes 22F and 33F.
  • composition in one embodiment includes capsular saccharides derived from serotypes 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (optionally conjugated).
  • at least 1 1 saccharide antigens are included, for example capsular saccharides derived from serotypes 1 , 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.
  • a vaccine may comprise capsular saccharides derived from serotypes 1 , 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F or capsular saccharides derived from serotypes 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F, although further saccharide antigens, for example 23 valent (such as serotypes 1 , 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by the invention.
  • 23 valent such as serotypes 1 , 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F
  • the antibody-mediated opsonic activity against the serotypes from which the at least 1 further saccharides are derived is also enhanced in the method, immunogenic composition or use of the present invention, in a further embodiment the opsonophagocytic response to 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 or 23 of the further Streptococcus pneumoniae capsular saccharides is enhanced.
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 1 saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 1 Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 4 saccharide (optionally wherein immunogenic composition enhances antibody-mediated opsonic activity against serotype 4 Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 5 saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 5 Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 5 saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 5 Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 5 saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 5 Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 5 saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 5 Streptococcus pneumoniae).
  • Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 6B saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 6B Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 7F saccharide (optionally wherein the immunogenic composition enhances antibody- mediated opsonic activity against serotype 7F Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 9V saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 9V Streptococcus
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 14 saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 14 Streptococcus pneumoniae). In a further embodiment the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 18C saccharide
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 19F saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 19F Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 23F saccharide (optionally wherein immunogenic composition enhances antibody- mediated opsonic activity against serotype 23F Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 3 saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 3 Streptococcus pneumoniae).
  • the 1 or more further Streptococcoccus pneumoniae saccharide conjugates comprises a conjugated serotype 19F saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 19F Strept
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 19A saccharide (optionally wherein the immunogenic composition enhances antibody-mediated opsonic activity against serotype 19A Streptococcus pneumoniae).
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates comprises a conjugated serotype 33F saccharide
  • carrier protein is intended to cover both small peptides and large polypeptides (>10 kDa).
  • the carrier protein may be any peptide or protein. It may comprise one or more T-helper epitopes.
  • the carrier protein may be tetanus toxoid (TT), tetanus toxoid fragment C, non-toxic mutants of tetanus toxin [note all such variants of TT are considered to be the same type of carrier protein for the purposes of this invention], polypeptides comprising tetanus toxin T-cell epitopes such as N19 (WO2006/067632), diphtheria toxoid (DT), CRM 197 (cross reacting material 197), other non-toxic mutants of diphtheria toxin [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida et al J. Biol. Chem.
  • CRM 9 CRM 45, CRM 102, CRM 103 and CRM 107 (where CRM stands for cross reacting material) and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel,
  • meningitidis serogroup B - EP0372501 meningitidis serogroup B - EP0372501
  • synthetic peptides EP0378881 , EP0427347
  • heat shock proteins WO 93/17712, WO 94/03208
  • pertussis proteins WO 98/58668, EP0471 177
  • cytokines, lymphokines, growth factors or hormones WO 91/01146
  • artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens (Falugi et al (2001 ) Eur J Immunol 31 ; 3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72; 4884-7) pneumococcal surface protein PspA (pneumococcal surface protein A) (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C.
  • H. influenzae Protein D (EP594610 and WO 00/56360), pneumococcal PhtA (Poly Histidine triad protein A) (WO 98/18930, also referred to Sp36), pneumococcal PhtD (Poly Histidine triad protein D (disclosed in WO 00/37105, and is also referred to Sp036D), pneumococcal PhtB (Poly Histidine triad protein B) (disclosed in WO 00/37105, and is also referred to Sp036B), or PhtE (Poly Histidine triad protein E) (disclosed in WO00/30299 and is referred to as BVH-3).
  • PhtA Poly Histidine triad protein A
  • Sp036D pneumococcal PhtD
  • PtB Poly Histidine triad protein B
  • PhtE Poly Histidine triad protein E
  • the Streptococcus pneumoniae capsular saccharide conjugate is conjugated to a carrier protein independently selected from the group consisting of tetanus toxoid (TT), fragment C of TT, diphtheria toxoid, CRM 197 (cross reacting material 197), detoxified pneumolysin, protein D (from H. influenzae), PhtD, PhtDE and N19.
  • TT tetanus toxoid
  • CRM 197 cross reacting material 197
  • detoxified pneumolysin protein D (from H. influenzae)
  • PhtD PhtD
  • PhtDE PhtDE
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates are independently conjugated to a carrier protein selected from the group consisting of tetanus toxoid (TT), fragment C of TT, diphtheria toxoid, CRM197, Pneumolysin, protein D, PhtD, PhtDE and N19.
  • TT tetanus toxoid
  • CRM197 tetanus toxoid
  • Pneumolysin protein D
  • PhtD PhtDE
  • N19 tetanus toxoid
  • the immunogenic composition comprises a saccharide from serotype 1 conjugated to protein D or CRM 197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 4 conjugated to protein D or CRM197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 5 conjugated to protein D or CRM 197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 6B conjugated to protein D or CRM197. In a further embodiment the composition comprises a saccharide from serotype 6B conjugated to protein D or CRM197.
  • the immunogenic composition comprises a saccharide from serotype 7F conjugated to protein D or CRM197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 9V conjugated to protein D or CRM 197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 14 conjugated to protein D or CRM197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 18C conjugated to Tetanus Toxoid or CRM197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 19F conjugated to Diphtheria Toxoid or CRM197.
  • the immunogenic composition comprises a saccharide from serotype 23F conjugated to protein D or CRM197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 3 conjugated to protein D or CRM197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 6A conjugated to protein D or CRM 197. In a further embodiment the immunogenic composition comprises a saccharide from serotype 19A conjugated to protein D or CRM 197.
  • a minority of the 1 or more further Streptocoocus pneumoniae saccharide conjugates are conjugated to protein D.
  • the term 'minority' refers to less than half.
  • 2, 3, 4 or 5 of the capsular saccharides from different serotypes are conjugated to protein D.
  • 2, 3, 4 or 5 of the capsular saccharides from different serotypes are conjugated to protein D.
  • 2, 3, 4 or 5 of the capsular saccharides from different serotypes are conjugated to protein D.
  • 2, 3, 4, 5 or 6 of the capsular saccharides from different serotypes are conjugated to protein D.
  • a 14 valent S. pneumoniae vaccine 2, 3, 4, 5, 6 or 7 of the capsular saccharides from different serotypes are conjugated to protein D.
  • a 15 valent S. pneumoniae vaccine 2, 3, 4, 5, 6 or 7 of the capsular saccharides from different serotypes are conjugated to protein D.
  • a 16 valent S. pneumoniae vaccine 2, 3, 4, 5, 6, 7 or 8 of the capsular saccharides from different serotypes are conjugated to protein D.
  • the immunogenic composition comprises between 1-20, 1-18, 1-16, -1-14, 1-12, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-2 Streptococcus pneumoniae capsular saccharide conjugated to protein D.
  • the immunogenic composition comprises at least one Streptococcus pneumoniae capsular saccharides conjugated to diphtheria toxoid. In a further embodiment the immunogenic composition comprises 19F conjugated to diphtheria toxoid. In one embodiment the immunogenic composition comprises at least one Streptococcus pneumoniae capsular saccharides conjugated to tetanus toxoid. In a further embodiment the immunogenic composition comprises 18C conjugated to tetanus toxoid.
  • the immunogenic composition comprises Streptococcus
  • pneumoniae conjugates comprising a serotype 1 saccharide conjugated to protein D, a serotype 4 saccharide conjugated to protein D, a serotype 5 saccharide conjugated to protein D, a serotype 6B saccharide conjugated to protein D, a serotype 7F saccharide conjugated to protein D, a serotype 9V saccharide conjugated to protein D, a serotype 14 saccharide conjugated to protein D, a serotype 23F saccharide conjugated to protein D, a serotype 18C saccharide conjugated to tetanus toxoid and a 19F saccharide conjugated to diphtheria toxoid.
  • the ratio of carrier protein to S. pneumoniae saccharide is between 1 :5 and 5: 1 ; 1 :2 and 2.5: 1 ; 1 : 1 and 2: 1 (w/w).
  • the majority of the conjugates, for example 6, 7, 8, 9 or more of the conjugates have a ratio of carrier protein to saccharide that is greater than 1 :1 , for example 1.1 : 1 , 1.2: 1 , 1.3: 1 , 1.4: 1 , 1.5: 1 or 1.6: 1.
  • saccharide throughout this specification may indicate polysaccharide or oligosaccharide and includes both.
  • Polysaccharides are isolated from bacteria and may be sized to some degree by known methods (see for example EP497524 and EP497525) and optionally by microfluidisation. Polysaccharides can be sized in order to reduce viscosity in polysaccharide samples and/or to improve filterability for conjugated products. Oligosaccharides have a low number of repeat units (typically 5-30 repeat units) and are typically hydrolysed polysaccharides.
  • Capsular polysaccharides of Streptococcus pneumoniae comprise repeating
  • oligosaccharide units which may contain up to 8 sugar residues.
  • a capsular saccharide antigen may be a full length polysaccharide, however in others it may be one oligosaccharide unit, or a shorter than native length saccharide chain of repeating oligosaccharide units. In one embodiment, all of the saccharides present in the vaccine are polysaccharides. Full length polysaccharides may be "sized" i.e. their size may be reduced by various methods such as acid hydrolysis treatment, hydrogen peroxide treatment, sizing by emulsiflex® followed by a hydrogen peroxide treatment to generate oligosaccharide fragments or microfluidization.
  • saccharide conjugates present in the immunogenic compositions of the invention may be conjugated to a carrier protein using any conjugation technique.
  • the Streptococcus pneumoniae saccharide is conjugated to the carrier protein via a linker, for instance a bifunctional linker.
  • the linker is optionally
  • heterobifunctional or homobifunctional having for example a reactive amino group and a reactive carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups.
  • the linker has for example between 4 and 20, 4 and 12, 5 and 10 carbon atoms.
  • a possible linker is ADH.
  • Other linkers include B-propionamido (WO 00/10599), 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).
  • ADH is used as a linker for conjugating saccharide from serotype 18C.
  • the saccharide conjugates present in the immunogenic compositions of the invention may be prepared by any known coupling technique.
  • the conjugation method may rely on activation of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.
  • CDAP 1-cyano-4-dimethylamino pyridinium tetrafluoroborate
  • the activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein.
  • the spacer could be cystamine or cysteamine to give a thiolated polysaccharide which could be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a haloacetylated carrier protein (for example using iodoacetimide [e.g. ethyl iodoacetimide HCI] or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP).
  • a maleimide-activated carrier protein for example using GMBS
  • a haloacetylated carrier protein for example using iodoacetimide [e.g. ethyl iodoacetimide HCI] or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP).
  • the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or ADH and the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the protein carrier.
  • carbodiimide e.g. EDAC or EDC
  • Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094.
  • This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group' reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate and coupling the CDI carbamate intermediate with an amino group on a protein.
  • the conjugates can also be prepared by direct reductive amination methods as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.
  • a further method involves the coupling of a cyanogen bromide (or CDAP) activated saccharide derivatised with adipic acid dihydrazide (ADH) to the protein carrier by Carbodiimide condensation (Chu C. et al Infect. Immunity, 1983 245 256), for example using EDAC(1-ethyl-2- (3-dimethylaminopropyl) carbodiimide).
  • a hydroxyl group optionally an activated hydroxyl group for example a hydroxyl group activated to make a cyanate ester [e.g.
  • a linker is present, a hydroxyl group on a saccharide is optionally linked to an amino group on a linker, for example by using CDAP conjugation.
  • a further amino group in the linker for example ADH) may be conjugated to a carboxylic acid group on a protein, for example by using carbodiimide chemistry, for example by using EDAC.
  • the pneumococcal capsular saccharide(s) is conjugated to the linker first before the linker is conjugated to the carrier protein.
  • the linker may be conjugated to the carrier before conjugation to the saccharide.
  • Carboxyl for instance via aspartic acid or glutamic acid.
  • this group is linked to amino groups on saccharides directly or to an amino group on a linker with
  • Amino group (for instance via lysine).
  • this group is linked to carboxyl groups on saccharides directly or to a carboxyl group on a linker with carbodiimide chemistry e.g. with EDAC.
  • this group is linked to hydroxyl groups activated with CDAP or CNBr on saccharides directly or to such groups on a linker; to saccharides or linkers having an aldehyde group; to saccharides or linkers having a succinimide ester group.
  • Sulphydryl for instance via cysteine.
  • this group is linked to a bromo or chloro acetylated saccharide or linker with maleimide chemistry.
  • this group is activated/modified with bis diazobenzidine.
  • protein carrier chemical group that may be generally used for coupling with a saccharide are amino groups (for instance on lysine residues), COOH groups (for instance on aspartic and glutamic acid residues) and SH groups (if accessible) (for instance on cysteine residues.
  • the modes of conjugation can be used to conjugate the Streptococcus pneumoniae capsular saccharide and/or the 1 or more further Streptococcus pneumoniae saccharides to a carrier protein.
  • the Streptococcus pneumoniae capsular saccharide is conjugated to a carrier protein through CDAP chemistry.
  • the 1 or more further Streptococcus pneumoniae saccharide conjugates are conjugated to a carrier protein through reductive amination.
  • Streptococcus pneumoniae saccharide conjugates are conjugated to a carrier protein through CDAP chemistry.
  • At least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 S. pneumoniae saccharides are conjugated to a carrier protein through reductive amination. In one embodiment less than 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 S. pneumoniae saccharides are conjugated to a carrier protein through reductive amination.
  • S. pneumoniae saccharide are conjugated to a carrier protein through reductive amination.
  • all of the S. pneumoniae capsular saccharides are conjugated to a carrier protein through reductive amination.
  • At least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 S. pneumoniae saccharides are conjugated to a carrier protein through CDAP chemistry. In one embodiment less than 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 S. pneumoniae saccharides are conjugated to a carrier protein through CDAP chemistry.
  • S. pneumoniae saccharide are conjugated to a carrier protein through CDAP chemistry. In a further embodiment all of the S.
  • the immunogenic composition of the invention comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 saccharides conjugated to a carrier protein through reductive amination and comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 saccharides conjugated to a carrier protein through a chemistry other than reductive amination for example CDAP chemistry.
  • capsular saccharides from at least one of the serotypes selected from the group consisting of serotypes 1 , 3, 19A and 19F are conjugated through a chemistry other than reductive amination and at least one of the serotypes selected from the group consisting of serotypes 4, 5, 6A, 6B, 6C, 7F, 9V, 14, 18C and 23F are conjugated through reductive amination.
  • the immunogenic composition of the invention comprises S.
  • the immunogenic composition of the invention comprises S.
  • 19F is conjugated to a carrier protein by CDAP chemistry.
  • pneumoniae capsular saccharide or group thereof is conjugated to a carrier protein by reductive amination; serotype 4, 5, 6A, 6B, 7F, 9V, 14, 18C or 23F, 4 and 5, 4 and 6A, 4 and 6B, 4 and 7F, 4 and 9V, 4 and 14, 4 and 18C, 4 and 23F, 5 and 6A, 5 and 6B, 5 and 7F, 5 and 9V, 5 and 14, 5 and 18C, 5 and 23F, 6A and 6B, 6A and 7F, 6A and 9V, 6A and 14, 6A and 18C, 6A and 23F, 6B and 7F, 6B and 9V, 6B and 14, 6B and 18C, 6B and 23F, 7F and 9V, 7F and 14, 7F and 18C, 7F and 23F, 9V and 14, 9V and 18C, 9V and 23F, 14 and 18C, 14 and 23F or 18C and 23F.
  • 23F is conjugated to a carrier protein by reductive amination
  • the immunogenic composition of the invention may comprise a dose of each saccharide conjugate between 0.1 and 20 ⁇ g, 1 and 5 ⁇ g, 1 and ⁇ g or 1 and 3 ⁇ g of saccharide per conjugate.
  • the immunogenic composition of the invention contains each S. pneumoniae capsular saccharide at a dose of between 0.1-20 ⁇ g; 0.5- ⁇ g; 0,5- 5 ⁇ g or 1 ⁇ g of saccharide.
  • capsular saccharides may be present at different dosages, for example some capsular saccharides may be present at a dose of exactly ⁇ g or some capsular saccharides may be present at a dose of exactly 3 ⁇ g.
  • the immunogenic composition comprises Streptococcus pneumoniae comprises capsular saccharide conjugates of saccharides from serotypes 1 , 5, 6B, 7F, 9V, 14 and 23F (and optionally 6A and/or 3) at dosages of ⁇ g of saccharide per conjugate.
  • saccharides from serotypes 3, 18C and 19F (or 4, 18C and 19F) are present at a higher dose than other saccharides.
  • serotypes 3, 18C and 19F (or 4, 18C and 19F) are present at a dose of around or exactly 3 ⁇ g whilst other saccharides in the immunogenic composition are present at a dose of around or exactly ⁇ g.
  • serotypes 1 , 5, 6B, 7F, 9V, 14 and 23F are present at a dose of around or exactly 1 ⁇ g.
  • the composition does not comprise an adjuvant comprising QS21 , Monophosphoryl lipid A (MPL), phospholipid and sterol, presented in the form of a liposome.
  • Compositions comprising adjuvant comprising QS21 , Monophosphoryl lipid A (MPL), phospholiopid and sterol presented in the form of a liposome are described in PCT application number WO2012/156391.
  • the immunogenic composition does not comprise an adjuvant comprising QS 21.
  • the immunogenic composition does not comprise an adjuvant comprising Monophosphoryl lipid A (MPL).
  • the immunogenic composition does not comprise either QS21 or MPL.
  • QS21 is an adjuvant derived from the tree Quillaja saponaria.
  • the immunogenic composition further comprises an adjuvant.
  • Suitable adjuvants include, but are not limited to, aluminium salts (aluminium phosphate or aluminium hydroxide), monophosphoryl lipid A (for example 3D-MPL), saponins (for example QS21 ), oil in water emulsions, blebs or outer membrane vesicle preparations from Gram negative bacterial strains (such as those taught by WO02/09746), lipid A or derivatives thereof, alkyl glucosamide phosphates or combinations of two or more of these adjuvants.
  • the adjuvant is aluminium phosphate.
  • the adjuvant comprises 100-750, 150-600, 200-500, 250-450, 300-400, or around 350 ⁇ g aluminium as aluminium phosphate per human dose.
  • the adjuvant is aluminium hydroxide.
  • the adjuvant does not comprise an oil in water adjuvant.
  • the present invention provides a vaccine comprising the immunogenic compositions of the invention.
  • Embodiments herein relating to "immunogenic compositions" of the invention are also applicable to embodiments relating to "vaccines” of the invention, and vice versa.
  • the vaccine comprises the immunogenic composition of the invention and a pharmaceutically acceptable excipient.
  • the vaccines of the invention may be administered by any suitable delivery route, such as intradermal, mucosal e.g. intranasal, oral, intramuscular or subcutaneous. Other delivery routes are well known in the art. 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).
  • the immunogenic composition of the invention is administered by the intramuscular delivery route.
  • Intramuscular administration may be to the thigh or the upper arm. Injection is typically via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used.
  • a typical intramuscular dose is 0.5 ml.
  • Intradermal administration of the vaccine forms an embodiment of the present invention.
  • Human skin comprises an outer "horny" cuticle, called the stratum corneum, which overlays the epidermis. Underneath this epidermis is a layer called the dermis, which in turn overlays the subcutaneous tissue.
  • the conventional technique of intradermal injection, the "mantoux procedure" comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow gauge needle (26 to 31 gauge) facing upwards the needle is inserted at an angle of between 10 to 15°. Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced whilst providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.
  • Alternative methods of intradermal administration of the vaccine preparations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (W099/27961 ), or transdermal patches (WO97/48440, WO98/28037), or applied to the surface of the skin (transdermal or transcutaneous delivery WO98/20734, WO98/28037).
  • the vaccine is in a low liquid volume, particularly a volume of between about 0.05 ml and 0.2 ml.
  • Another suitable administration route is the subcutaneous route.
  • Any suitable device may be used for subcutaneous delivery, for example classical needle.
  • a needle-free jet injector service is used, such as that published in WO01/05453, WO01/05452, WO01/05451 , WO01/32243, WO01/41840, WO01/41839, WO01/47585, WO01/56637, WO01/58512, WO01/64269, WO01/78810, WO01/91835, WO01/97884, WO02/09796, WO02/34317.
  • the device is pre-filled with the liquid vaccine formulation.
  • the vaccine is administered intranasally.
  • the vaccine is administered locally to the nasopharyngeal area, e.g. without being inhaled into the lungs.
  • an intranasal delivery device which delivers the vaccine formulation to the nasopharyngeal area, without or substantially without it entering the lungs.
  • Preferred devices for intranasal administration of the vaccines according to the invention are spray devices. Suitable commercially available nasal spray devices include AccusprayTM (Becton Dickinson).
  • spray devices for intranasal use are devices for which the
  • intranasal devices produce droplets (measured using water as the liquid) in the range 1 to 200 ⁇ , e.g. 10 to 120 ⁇ .
  • Bi-dose delivery is another embodiment of an intranasal delivery system for use with the vaccines according to the invention.
  • Bi-dose devices contain two sub-doses of a single vaccine dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time.
  • a monodose device may be used for administering the vaccines according to the invention.
  • a further aspect of the invention is a method of making a vaccine of the invention comprising the steps of mixing the unconjugated S. pneumoniae protein with the adjuvant composition.
  • the vaccine of the invention may be administered as a single dose
  • components thereof may also be co-administered together at the same time or at different times (for instance pneumococcal saccharide conjugates could be administered separately, at the same time or 1 to 2 weeks after the administration of the any bacterial protein component of the vaccine for optimal coordination of the immune responses with respect to each other).
  • subjects may receive one or several booster immunisation adequately spaced.
  • Around or “approximately” are defined as within 10% more or less of the given figure for the purposes of the invention.
  • dPly-10-AIPO 4 A vaccine comprising 1C ⁇ g of dPIy adjuvanted with Aluminium phosphate.
  • dPly-30-AIPO 4 A vaccine comprising 3C ⁇ g of dPIy adjuvanted with Aluminium phosphate.
  • dPly/PhtD-10-AIPO 4 A vaccine comprising 1 C ⁇ g of dPIy and 1 C ⁇ g of PhtD adjuvanted with Aluminium phosphate.
  • dPly/PhtD-30-AIPO 4 A vaccine comprising 3C ⁇ g of dPIy and 3C ⁇ g of PhtD adjuvanted with Aluminium phosphate.
  • 10PCV/dPly/PhtD -10-AIPO 4 A vaccine comprising the following antigens adjuvanted to Aluminium phosphate:
  • 10PCV/dPly/PhtD -30-AIPO 4 A vaccine comprising the following antigens adjuvanted to Aluminium phosphate:
  • 10PCV A vaccine comprising the following antigens adjuvanted to Aluminium phosphate:
  • Pneumococcal pneumolysin was prepared and detoxified as described WO2004/081515 and WO2006/32499 using formaldehyde detoxification.
  • PhtD protein is a member of the pneumococcal histidine-triad (Pht) protein family characterized by the presence of histidine-triads.
  • PhtD is a 838 aa- molecule and carries 5 histidine triads (see Medlmmune WO00/37105 SEQ ID NO: 4 for amino acid sequence and SEQ ID NO: 5 for DNA sequence).
  • PhtD also contains a proline-rich region in the middle (amino acid position 348-380). PhtD has a 20 aa-N-terminal signal sequence.
  • PhtD is described in WO2007/071710 (see for example Example 1 b).
  • Protein D was expressed as described in WO2007/071710
  • polysaccharides may be sized by microfluidisation as described below.
  • the activation and coupling conditions are specific for each polysaccharide. These are given in Table 1. Sized polysaccharide (except for 6B and 23F) was dissolved in NaCI 2M, NaCI 0.2M or in water for injection (WFI). The optimal polysaccharide concentration was evaluated for all the serotypes. All serotypes except serotype 18C were conjugated directly to the carrier protein as detailed below.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • CDAP/PS ratio 0.5-1.5 mg/mg PS
  • Purified protein (protein D, CRM 197 or DT) (the quantity depends on the initial PS/carrier protein ratio) was added to the activated polysaccharide and the coupling reaction was performed at the specific pH for up to 2 hour (depending upon serotype) under pH regulation. In order to quench un-reacted cyanate ester groups, a 2M glycine solution was then added to the mixture. The pH was adjusted to the quenching pH (pH 9.0). The solution was stirred for 30 minutes at 25 °C and then incubated overnight at 2-8 °C with continuous slow stirring.
  • Polysaccharide serotype 18C was microfluidized before conjugation. Derivatization of tetanus toxoid with EDAC (1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide)
  • purified TT was diluted at 25 mg/ml in 0.2M NaCI and the ADH spacer was added in order to reach a final concentration of 0.2M.
  • the pH was adjusted to 6.2.
  • EDAC 1-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide
  • Derivatized TT was then diafiltrated (10 kDa CO membrane) in order to remove residual ADH and EDAC reagent.
  • CDAP solution 100 mg/ml freshly prepared in 50/50 v/v acetonitrile/WFI was added to reach the appropriate CDAP/PS ratio.
  • the pH was raised up to the activation pH 9.0 by the addition of 0.3M NaOH and was stabilised at this pH until addition of TT AH .
  • the pH was adjusted to the quenching pH (pH 9.0).
  • the solution was stirred for 30 min at 25 °C, and then overnight at 2-8°C with continuous slow stirring.
  • CDAP cone 0.50 0.75 0.75 1.5 1.5 0.79
  • pHa,c,q corresponds to the pH for activation, coupling and quenching, respectively
  • the conjugates were purified by gel filtration using a Sephacryl S400HR gel filtration column equilibrated with 0.15M NaCI (except S500HR was used as buffer for 18C and 20mM acetate containing 1.15MNaCI pH6.2 was used for 19A) to remove small molecules (including DMAP) and unconjugated saccharide and protein.
  • PS-PD, PS-TT, PS-CRM 197 or PS-DT conjugates are eluted first, followed by free PS, then by free protein carrier and finally DMAP and other salts (NaCI, glycine).
  • dPly-10-AIPO 4 group subjects receiving the dPly-10- ALP0 4 vaccine as described in example 1 (formulated with 167 ⁇ g AIP0 4 , 150mM NaCI and 0.78mM P0 4 buffer).
  • dPly-30-AIPO 4 group subjects receiving the dPly-30- ALPO 4 vaccine as described in example 1 (formulated with 500 AIP0 4, 150nM NaCI and 0.78mM P0 4 buffer).
  • dPly/PhtD-10-AIPO 4 group subjects receiving the dPly/PhtD-10-AIPO 4 vaccine as described in example 1 (formulated with 500 ⁇ g AIP0 4, 150mM NaCI and 0.72mM P0 4 buffer).
  • dPly/PhtD-30-AIPO 4 group subjects receiving the dPly/PhtD-30-AIPO 4 vaccine as described in example 1 (formulated with 500 ⁇ g AIP0 4, 150mM NaCI and 0.86mM P0 4 buffer).
  • 10PCV/dPly/PhtD -10-AIPO 4 group ( ⁇ 0 ⁇ 10' in result tables and figures): subjects receiving the 10PCV/dPly/PhtD -I O-AIPO 4 vaccine as described in example 1 (formulated with 500 ⁇ g AIP0 4, 150mM NaCI and 0.72mM P0 4 buffer).
  • 10PCV/dPly/PhtD -30 -AIP0 4 group ( ⁇ 0 ⁇ 30' in result tables and figures): subjects receiving the 10PCV/dPly/PhtD -30-AIPO 4 vaccine as described in example 1 (formulated with 500 ⁇ g AIP0 4, 150mM NaCI and 0.86 mM P0 4 buffer).
  • 23PPV Comparator group subjects receiving one dose of a commercially available licensed 23-valent c Polysacharide Vaccine (23PPV) named Pneumovax 23TM at Day 0 (Month 0) followed by placebo at Day 60 (Month 2).
  • 23PPV 23-valent c Polysacharide Vaccine
  • Vaccines were administered as an intramuscular injection in the non dominant deltoid.
  • Subjects were healthy males or females between, and including, 18 and 40 years old at the time of the first vaccination.
  • Regulatory Activities with 95% CI (confidance interval) according to the intensity and relationship to vaccination. Prevalence of concomitant antipyretic/medication during the 7-day post-vaccination follow-up period and during the 31 -day post-vaccination follow-up period was computed with 95% CI, after each vaccine dose and overall. Serious adverse events (SAEs) and withdrawals due to adverse event(s) reported during the entire study period were described in detail. For each haematology and biochemistry parameter, the number and percentage of subjects with levels below, within and above normal ranges were tabulated per study group, at each assessed time point.
  • SAEs Serious adverse events
  • the grading and the change in grading from baseline was summarized per study group, at each assessed time point.
  • For each urine parameter the grading and the change in grading from baseline (baseline: day 0 assessment) was summarized per study group, at each assessed time point. pH quantified in urinalysis over time was displayed per study group.
  • Opsonophagocytic activity for antibodies against each of the 10 pneumococcal serotypes was measured by a killing-assay using a HL 60 cell line. The results were presented as the dilution of serum
  • opsonic titre able to sustain 50% killing of live pneumococci under the assay conditions.
  • the cut-off of the assay was an opsonic titre of 8.
  • Anti-dPly and anti-PhtD antibodies were quantified using an ELISA (enzyme linked immunosorbent assay).
  • ELISA enzyme linked immunosorbent assay
  • Microtiter plates are coated with purified serotype specific pneumococcal PS (polysaccharide) and mixed with methylated human serum albumin (0.5 to 7.5 g/ml, depending on the PS). Serum samples are diluted in adsorption buffer (phosphate-buffered saline [PBS], 10% fetal calf serum [FCS], 0.1 % polyvinyl alcohol) containing 10 ⁇ g/ml of the CWPS (capsular wall polysaccharide) (SSI) and 2 ⁇ g/ml of PS of serotype 22F (ATCC) and are incubated overnight at 4°C.
  • adsorption buffer phosphate-buffered saline [PBS], 10% fetal calf serum [FCS], 0.1 % polyvinyl alcohol
  • CWPS capsule wall polysaccharide
  • ATCC serotype 22F
  • the 89-SF reference serum sample (FDA) (89-SF is the number of the reference serum calibrated by the World Health Organisation) is diluted in the same adsorption buffer but with 10 ⁇ g/ml of CWPS only, and the mixture is incubated overnight at 4°C.
  • the 89-SF reference serum sample is preadsorbed only with CWPS and not with 22F because the values for the anti- PS concentrations of the 89-SF reference serum sample are assigned without the use of 22F PS. Bound antibodies are detected by using alkaline phosphatase-conjugated anti-human IgG.
  • OPA opsonophagocytosis assay
  • the OPA assay is performed as follows: serum samples are heated for 40 min at 56 °C to inactivate any remaining endogenous complement. Twenty-five microlitres aliquots of each 1 :2 diluted serum sample is two-fold serially diluted in 25 ⁇ OPA buffer (Hank's Balanced salt solution or HBSS -14.4% inactivated FBS (fetal bovine serum)) per well of a 96- well round bottom microtitre plate (Nalge Nunc International, Rochester, NY, USA). Two positive control samples with known opsonophagocytic titre for the specific pneumococcal serotype are run on each assay plate to determine the assay validity.
  • the assay plate is incubated for 2 h at 37 °C with orbital shaking (210 rpm) to promote the phagocytic process.
  • the reaction is stopped by laying the microplate on ice for at least 1 min.
  • a 20 ⁇ aliquot of each well of the plate is then transferred into the corresponding well of a 96-well flat bottom microplate (Nalge Nunc International) and 100 ⁇ of Todd-Hewitt Broth-0.9% agar is added to each well.
  • pneumococcal colonies appearing in the agar are counted using an automated image analysis system (KS 400, Zeiss, Oberkochen,
  • the OPA titre for the serum samples is determined by the reciprocal dilution of serum able to facilitate 50% killing of the pneumococci.
  • the opsonophagocytic titre is calculated by using a 4-parameter curve fit analysis. Each sample titre was adjusted through an adjustment factor based on the average ratio of actually measured titre/known titre of two positive control samples included in each assay.
  • the cut-off OPA titre for all serotypes is a reciprocal serum dilution of 8. Serum samples with OPA titres ⁇ 8 are assigned a titre of 4 for the purpose of data analysis.
  • the plates are saturated for 1 hour at RT with 200 ⁇ / ⁇ 8 of diluant buffer (PBS without Ca and Mg -BSA 1 % for PhtD) or BSA 5% for Ply - Polysorbate 20 0.1 % - ProClin 300TM 0.2% pH 7.0)with shaking.
  • diluant buffer PBS without Ca and Mg -BSA 1 % for PhtD
  • the plates are incubated for 1 hour at room temperature with shaking. After 5 washes, the plates are incubated with a goat anti -human IgG Fc antibody conjugated to peroxydase , diluted 1/20000 for PhtD and 1/12500 for Ply (100 ⁇ per well) at room temperature for 1 hour with shaking. Plates are washed as above and 100 ⁇ of the TMB substrate conjugate is added to each well for 10 min in darkness.
  • the reaction is stopped by addition of H2S04 (0.18M) 100 ⁇ and the absorbance is read at 450nm and 650 nm.
  • Immunogenicity analysis was performed on the according to protocol (ATP) cohort for immunogenicity. No second analysis on the Total vaccinated cohort was performed as less than 5% of subjects were excluded from the ATP cohort for immunogenicity. Sera were analysed for the presence of antibodies against dPIy, PhtD, and Streptococcus pneumoniae capsular saccharide serotypes 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.
  • a seropositive subject was defined as a subject with antibody concentration > 0.05 ⁇ 9/ ⁇ _.
  • GMC geometric mean antibody concentration calculated on all subjects
  • N number of subjects with available results
  • n/% number/percentage of subjects with concentration within the specified range
  • PI(D30) blood sample taken post dose I at day 30
  • PI I (D90) blood sample taken post dose I I at day 90
  • PI(D30) 24 24 100 85.8 100 12903.5 9567.7 17402.4
  • Anti-PhtD antibody GMCs post-dose 1 and post dose-2 tended to be lower in the 10vPP10 and 10vPP30 groups compared to the
  • Seropositivity rates and GMTs for serotype 1 antibodies are described in Table 3 below, these data are illustrated in Figure 3: Table 3: Seropositivity rates and GMTs for anti-PS1 antibodies (ATP cohort for immunogenicity)
  • Table 8 Seropositivity rates and GMTs for anti-PS9V antibodies (ATP cohort for immunogenicity)
  • Seropositivity rates and OPA GMTs for serotype 14 antibodies are described in Table 9 below, these data are illustrated in Figure 9: Table 9: Seropositivity rates and GMTs for anti-PS14 antibodies (ATP cohort for immunogenicity)
  • PI(D30) 24 24 100 85.8 100 3058.8 1829.4 51 14.2 PII(D90) 22 22 100 84.6 100 2551.9 1518.7 4287.9
  • Table 11 Seropositivity rates and GMTs for anti-PS19F antibodies (ATP cohort for
  • the formulations containing the pneumococcal protein dPIy with or without PhtD were immunogenic. Increases in the anti-Ply antibody GMCs were observed following each vaccination in the groups vaccinated with dPIy containing formulations; increase in anti- PhtD antibody GMCs were observed following each vaccination in groups vaccinated with PhtD containing formulations. The highest anti-Ply and anti-PhtD antibody GMCs were observed for the formulations containing 30 ⁇ g of dPIy and PhtD proteins (i.e in the group PIPh-30 followed by 10vPP30 group).
  • Free protein formulations do not induce an OPA responses
  • One month post-dose 1 and one month post-dose 2 for each of the vaccine pneumococcal serotypes, at least 95.7% of subjects receiving the formulation combining pneumococcal proteins (dPIy and PhtD) and 10Pn-PD-DiT had OPA titres ⁇ 8.
  • An increase in OPA GMTs was observed from post-dose 1 to post-dose 2 timepoint for some of the pneumococcal serotypes.
  • the calculated OPA GMT point estimates post dose 1 or post dose 2 were higher in the 10vPP30 groups as compared to the 10vPP10 group.
  • Example 3 Clinical trial to study efficacy of vaccines comprising high and low doses of dPIy and PhtD carried out in infants
  • 10PCV/dPly/PhtD-30-AIPO 4 group ( ⁇ 0 ⁇ 30' in result tables and figures): subjects who received GSK Biologicals' 10PCV/dPly/PhtD-30-AIPO 4 vaccine co-administered with DTPa-HBV-IPV/Hib vaccine.
  • Prev13 group subjects who received Pfizer's commercially available 13-valent pneumococcal conjugate vaccine (Prevenar 13) coadministered with DTPa-HBV-IPV/Hib vaccine.
  • the vaccines were given to healthy infants according to a 3-dose primary vaccination schedule at 2, 3, 4 months of age (Epoch 1 , completed) to be followed by a booster dose at 12-15 months of age (Epoch 2, study ongoing).
  • the percentage of subjects reporting each individual solicited local and general AE (adverse event) during the 7-day (Day 0-Day 6) solicited follow-up period were tabulated for each group, after each vaccine dose and overall primary doses, with exact 95% CI (confidance interval).
  • the percentage of doses followed by each individual solicited local and general AE was tabulated for each group, over the full primary vaccination course, with exact 95% CI.
  • the same tabulations were performed for grade 3 solicited AEs and for solicited AEs with causal relationship to vaccination. For redness and swelling, grade 2 or 3 AEs were also tabulated. Occurrence of fever was reported per 0.5°C cumulative increments. All the above tabulations for each individual solicited adverse event were also performed for the 4-day follow-up period after each vaccination (Day 0-Day 3).
  • the proportion of subjects/doses with at least one report of unsolicited AE classified by the Medical Dictionary for Regulatory Activities (MedDRA) and reported up to 30 days after primary vaccination was tabulated with exact 95% CI for each group. The same tabulation was performed for grade 3 unsolicited AEs and for unsolicited AEs with a relationship to vaccination.
  • Pneumococcal serotype specific total IgG antibodies (antibodies to 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) were each measured by 22F-inhibition ELISA (enzyme-linked immunosorbent assay) as described for example 2.
  • the antibody concentration was determined by logistic log comparison of the ELISA curves with a standard reference serum 89-SF available from the US Food and Drug Administration (FDA) for which concentration of IgG and IgM to the 10 serotypes are known in ⁇ g/mL.
  • the cut-off of the assay was 0.05 ⁇ g/mL.
  • Opsonophagocytic activity for antibodies against each of the
  • pneumococcal serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F was measured by an OPA assay (similar to that described in example 2).
  • OPA assay similar to that described in example 2.
  • the results were presented as the dilution of serum (opsonic titre) able to sustain 50% killing of live pneumococci under the assay conditions.
  • the cut-off of the assay was an opsonic titre of 8.
  • Anti-Ply and anti-PhtD antibodies were quantified using individual ELISA.
  • Concentration of specific anti-Ply and anti-PhtD antibodies was determined using a standard reference serum. The cut-off of the assay was 100 EL.U/mL for PD, 12 EL.U/mL for dPIy and 17 EL.U/mL for PhtD.
  • Redness was the most frequently reported solicited local AE in each group (after 43.6%, 44.7%, 40.1 % and 41.1 % of doses in the 10PCV/dPly/PhtD -10-AIPO 4 , 10PCV/dPly/PhtD -30-AIPO 4 , 10PCV and Prev13 groups, respectively). No more than 3.3% of doses were followed by grade 3 solicited local AEs of given category, in each group.
  • Irritability was the most frequently reported solicited general AE in each group (after 55.5%, 55.5%, 55.0% and 56.6% of doses in the 10PCV/dPly/PhtD -10- AIP0 4 , 10PCV/dPly/PhtD -30-AIPO 4 , 10PCV and Prevl 3 groups, respectively). No more than 4.8% of doses were followed by grade 3 general solicited AEs of given category, in each group and most of them were considered by the investigator to be causally related to vaccination.
  • Grade 3 unsolicited AEs were reported after 0.0%, 0.2%, 0.5% and 0.2% of doses in the 10PCV/dPly/PhtD -10-AIPO 4 , 10PCV/dPly/PhtD -30-AIPO 4 , 10PCV and Prevl 3 group, respectively. Of these, one grade 3 AE (hypotonic hyporesponsive episode) reported in the 10PCV group was considered by the investigator to be causally related to vaccination.
  • Example 4 Formulation of multivalent vaccines comprising a PE-PilA fusion protein
  • 10V A ten valent (10V) vaccine containing the following ten S. pneumoniae capsular saccharide conjugates: capsular saccharide from serotype 1 conjugated to protein D (1-PD), capsular saccharide from serotype 4 conjugated to protein D (4-PD), capsular saccharide from serotype 5 conjugated to protein D (5-PD), capsular saccharide from serotype 6B conjugated to protein D (6B-PD), capsular saccharide from serotype 7F conjugated to protein D (7F-PD), capsular saccharide from serotype 9V conjugated to protein D (9V-PD), capsular saccharide from serotype 14 conjugated to protein D (14-PD), capsular saccharide from serotype 23F conjugated to protein D (23F-PD), capsular saccharide from serotype 18C conjugated to tetanus toxoid (18C-TT) and capsular saccharide from serotype 19F conjugated to Diphtheria Toxin (19F-DT).
  • 12V A twelve valent (12V) vaccine containing the same ten S. pneumoniae capsular saccharide conjugates as 10V with an additional two S. pneumoniae saccharide conjugates, 19A conjugated to CRM197 (19ACRM) and 6A conjugated to CRM197 (6ACRM).
  • 12V+ proteins (12V+prot): A vaccine containing the same twelve S. pneumoniae capsular saccharide conjugates as 12V with the addition of PhtD, dPIy and PE-PilA fusion protein. Preparation of the antigens
  • the antigens were prepared as described in example 1.
  • the PEPilA antigen was prepared as described in WO2012/139225.
  • the 10V vaccine contains S. pneumoniae capsular saccharide serotype 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugates adsorbed onto aluminium phosphate together at a human dose of 1 , 3, 1 , 1 , 1 , 1 , 1 , 3, 3, 1 (the saccharides were individually adsorbed to aluminium
  • the 12V vaccine was made in the same way as the 10V vaccine with additional serotypes 19A and 6A conjugates at doses of 2 ⁇ g adsorbed onto aluminium phosphate added.
  • the 12V + proteins vaccine was made in the same way as the 12V vaccine except proteins PhtD, dPIy and PE-PilA were added.
  • the 12 conjugates and the proteins were mixed together using the dosages described for 12V above and the proteins at dosages of 30 ⁇ g each (note that this refers to 30 ⁇ g of PE-PilA, not 30 ⁇ g of PE and 30 ⁇ g of PilA).
  • Example 5 Comparison of the immunoqenicitv of 10V, 12V and 12V+proteins vaccines in mice
  • Sera was diluted (1/2 for 6A and 6B and 1/10 for the other serotypes) in PBS-Polysorbate20 0.05% containing CPS (1 mg CPS/ml of non diluted serum except or 6A and 6B which was at 2.5mg/ml) V/V and incubated for 1 hour at 37°C in order to neutralize antibodies directed to the CPS.
  • the plates were incubated under agitation for 30 minutes at room temperature. The plates were washed as above and anti-mouse IgG antibodies conjugated to peroxidase (100 ⁇ per well) was added, the plates were incubated for 30 minutes at room temperature with shaking. After washing, the substrate (4 mg of OPDA (ortho phenylen-diamine) in 10 ml of citrate 0.1 M pH 4.5- 4.6 and 5 ⁇ of H 2 0 2 ) was added to each well (100 ⁇ ) and the plates incubated for 15 minutes in the dark. The reaction was stopped by addition of HCI 1 N (50 ⁇ ). The absorbance was read at 490nm or 620nm for the reference using a spectrophotometer. The color developed is directly proportional to the amount of antibody present in the serum.
  • OPDA ortho phenylen-diamine
  • mice immunised as described in the section entitled 'immunogenicity of three vaccine formulations in mice' or a reference serum (an internal reference calibrated with Chrompure mouse IgG) was added to microwells and serially diluted 100 ⁇ (two-fold dilution step) in PBS Polysorbate 20 0.05% (for the PD assay) and PBS Polysorbate 20 0.05% BSA 0.1 % (for the PE and PilA assay).
  • PBS Polysorbate 20 0.05% for the PD assay
  • PBS Polysorbate 20 0.05% BSA 0.1 % for the PE and PilA assay
  • the plates were then washed as above and the substrate conjugate (4 mg of OPDA (ortho phenylen-diamine) in 10 ml of citrate 0.1 M pH 4.5-4.6 and 5 ⁇ of H 2 0 2 ) as added to each well (100 ⁇ ) for 15 min in darkness.
  • the reaction was stopped by addition of HCI 1 N 50 ⁇ and the absorbance was read at 490 nm (620 nm for the reference).
  • the plates were washed as above and the substrate conjugate (4 mg of OPDA in 10 ml of citrate 0.1 M ph 4.5 and 5 ⁇ of H 2 0 2 ) was added to each well (100 ⁇ ) for 15 min in darkness. The reaction was stopped by addition of HCI 1 N 50 ⁇ and the absorbance was read at 490 nm (620 nm for the reference filter).
  • Serum samples were heated for 45 min at 56 °C to inactivate any remaining endogenous complement. Twenty-five microlitres aliquots of each 1 :2 diluted serum sample were two-fold serially diluted in 25 ⁇ OPA buffer (HBSS (Hank's Balanced Salt Solution)-14.4% inactivated FCS (Foetal Calf Serum)) per well of a 96-well round bottom microtitre plate. Subsequently, 25 ⁇ of a mixture of activated HL-60 cells (1 ⁇ 107 cells/ml), freshly thawed pneumococcal working seed and freshly thawed baby rabbit complement in an e.g.
  • OPA buffer HBSS (Hank's Balanced Salt Solution)-14.4% inactivated FCS (Foetal Calf Serum)
  • pneumococcal colonies which appeared in the agar were counted using an automated image analysis system (KS 400, Zeiss, Oberkochen, Germany). Eight wells without serum sample were used as bacterial controls to determine the number of pneumococci per well. The mean number of CFU of the control wells was determined and used for the calculation of the killing activity for each serum sample.
  • the OPA titre for the serum samples was determined by the reciprocal dilution of serum able to facilitate 50% killing of the pneumococci.
  • the opsonophagocytic titre was calculated by using a 4-parameter curve fit analysis.
  • Immunoqenicity of three vaccine formulations in mice 2 groups of 27 female Balb/c mice were immunized by intramuscular (IM) injections on days 0, 14 and 28 with 1/10 human dose of different formulation including Proteins alone (PhtD, dPIy and PEPilA - results not presented), Prevnar 13 ( T M a commercially available Streptococcal vaccine - results not presented) 10V, 12V (DSP2A017) and 12V+ proteins (DSP2A012) GMP lots.
  • IM intramuscular
  • mice received in a different leg 1/10 th human dose of Infanrix Hexa ( T M a vaccine comprising diphtheria toxoid, tetanus toxoid, pertussis toxoid, filamentous haemmagglutinin, pertactin, hepatitis B surface antigen, inactivated polio virus types 1 , 2 and 3 and Haemophilus influenzae b saccharide (PRP)).
  • T M a vaccine comprising diphtheria toxoid, tetanus toxoid, pertussis toxoid, filamentous haemmagglutinin, pertactin, hepatitis B surface antigen, inactivated polio virus types 1 , 2 and 3 and Haemophilus influenzae b saccharide (PRP)).
  • T M a vaccine comprising diphtheria toxoid, tetanus toxoid, pertussis to
  • Anti-lgG levels and opsonophagocytosis titers were determined respectively in individual and pooled sera collected at day 42.
  • the potential of the 12V +proteins vaccine to induce IgG antibody titers and opsonic activity was evaluated and compared to that of the 12V and 10V vaccines.
  • Example 6 Comparison of the immunoqenicitv of 10V, 12V and 12V+ protein vaccines in guinea pigs
  • Microplates were coated for 2 hours at 37°C with 100 ⁇ per well of 2.5 ⁇ g/ml of PS1 , 5 ⁇ g/ml of PS4, 5, 6A, 6B, 7F, 9V or 14; 10 ⁇ g/ml of PS19A and 23F, 40 ⁇ g/ml of PS18C and PS19F or Affinipure Goat anti-guinea pig lgG(2.4mg/ml) diluted to 2 ⁇ g/ml for the reference wells in PBS.
  • the plates were washed three times with NaCI 150 mM (0.9%)-Polysorbate20 0.05%.
  • the serum from guinea pigs immunised as described in the section entitled 'immunogenicity of three vaccine formulations in guinea pigs' was added to the microwells and serially diluted 100 ⁇ (two-fold dilution step) in PBS-Polysorbate20 0.05% or a reference serum (Chrompure guinea pig IgG (1 1 mg/ml) diluted to 0.25 ⁇ g/ml in PBS-polysorbate20 0.05%) was added . The plates were incubated under agitation for 30 minutes at room temperature.
  • the plates were washed as above and anti-guinea pig IgG antibodies conjugated to peroxidase (100 ⁇ per well) were added and the plates incubated for 30 minutes at room temperature with shaking. After washing, the substrate (4 mg of OPDA in 10 ml of citrate 0.1 M pH 4.5-4.6 and 5 ⁇ of H 2 0 2 ) was added to each well (100 ⁇ ) and incubated for 15 minutes in the dark. The reaction was stopped by addition of HCI 1 N. Absorbance was read at 490nm (620nm for the reference) using a spectrophotometer. The color developed is directly proportional to the amount of antibody present in the serum.
  • Plates were coated for 2 hours at 37°C with 100 ⁇ per well of 2 ⁇ g/ml of PD (1 mg/ml), 2 ⁇ g/ml of PE (1500 g/ml), or 2 g/ml of PilA (3660 g/ml) in carbonate buffer pH 9.6 in PBS or Affinipure Goat anti-guinea pig IgG (2.4mg/ml) diluted to 2 ⁇ g/m for the reference wells in PBS. The plates were washed 4 times with NaCI 0.9% Polysorbate 20 0.05%. For PE and PilA ELISAs (this step was not carried out for the PD and Ply ELISAs), the plates were saturated 30 min at room temperature with PBS-BSA1 %.
  • the plates were incubated with an anti-guinea pig IgG antibody conjugated to peroxydase (100 ⁇ per well) at room temperature for 30 minutes with shaking. Plates were washed as above and the substrate conjugate (4 mg of OPDA in 10 ml of citrate 0.1 M pH 4.5-4.6 and 5 ⁇ of H 2 0 2 ) was added to each well (100 ⁇ ) for 15 min in darkness. The reaction was stopped by addition of HCI 1 N 50 ⁇ and the absorbance is read at 490 nm (620 nm for the reference filter). Description of the ELISAto measure PhtD and dPIv antibodies
  • the plates were incubated for 30 min at room temperature with agitation. After washing, the plates were incubated with an anti-guinea pig IgG antibody conjugated to peroxydase (100 ⁇ per well) at room temperature for 30 minutes with shaking. Plates were washed as above and the substrate conjugate (4 mg of OPDA in 10 ml of citrate 0.1 M pH 4.5-4.6 and 5 ⁇ of H 2 0 2 ) was added to each well (100 ⁇ ) for 15 min in darkness. The reaction was stopped by addition of HCI 1 N 50 ⁇ and the absorbance was read at 490 nm (620 nm for the reference filter).
  • Serum samples were heated for 45 min at 56 °C to inactivate any remaining endogenous complement. Twenty-five microlitres aliquots of each 1 :2 diluted serum sample was two-fold serially diluted in 25 ⁇ OPA buffer (HBSS (Hank's Balanced Salt Solution)-14.4% inactivated FCS (foetal calf serum)) per well of a 96-well round bottom microtitre plate. Subsequently, 25 ⁇ of a mixture of activated HL-60 cells (1 ⁇ 107 cells/ml), freshly thawed pneumococcal working seed and freshly thawed baby rabbit complement in an e.g.
  • OPA buffer HBSS (Hank's Balanced Salt Solution)-14.4% inactivated FCS (foetal calf serum)
  • pneumococcal colonies appearing in the agar were counted using an automated image analysis system (KS 400, Zeiss, Oberkochen, Germany). Eight wells without serum sample were used as bacterial controls to determine the number of pneumococci per well. The mean number of CFU of the control wells was determined and used for the calculation of the killing activity for each serum sample.
  • the OPA titre for the serum samples was determined by the reciprocal dilution of serum able to facilitate 50% killing of the pneumococci.
  • the opsonophagocytic titre was calculated by using a 4-parameter curve fit analysis.
  • T M a vaccine comprising diphtheria toxoid, tetanus toxoid, pertussis toxoid, filamentous haemmagglutinin, pertactin, hepatitis B surface antigen, inactivated polio virus types 1 , 2 and 3 and Haemophilus influenzae b saccharide (PRP).
  • Anti-lgG levels and opsonophagocytosis titers were determined respectively in individual and pooled sera collected at days 42.
  • the IgG antibody titers and opsonic activity was evaluated and compared between the
  • Sera from guinea pigs injected with the different formulations were tested in ELISA against the polysaccharide serotypes and proteins and in OPA against the 12 serotypes in the formulation.
  • the 12V + proteins induced similar responses to the 12V formulation.

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Abstract

La présente invention concerne une composition immunogène comprenant (i) au moins une protéine de Streptococcus pneumoniae; (ii) un saccharide capsulaire de Streptococcus pneumoniae dérivé d'une souche de Streptococcus pneumoniae d'un sérotype ciblé, ladite composition étant destinée à être utilisée pour augmenter l'activité opsonique médiée par les anticorps contre le sérotype ciblé de Streptococcus pneumoniae.
EP13776808.1A 2012-10-17 2013-10-15 Composition immunogène Ceased EP2908854A2 (fr)

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US201261714942P 2012-10-17 2012-10-17
US201261714956P 2012-10-17 2012-10-17
GBGB1218660.7A GB201218660D0 (en) 2012-10-17 2012-10-17 Immunogenic composition
US13/826,696 US9561268B2 (en) 2012-10-17 2013-03-14 Immunogenic composition
US13/827,203 US20140105927A1 (en) 2012-10-17 2013-03-14 Immunogenic composition
US13/826,932 US20140193451A1 (en) 2012-10-17 2013-03-14 Immunogenic composition
PCT/EP2013/071483 WO2014060389A2 (fr) 2012-10-17 2013-10-15 Composition immunogène

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EP13779190.1A Active EP2908855B1 (fr) 2012-10-17 2013-10-15 Composition immunogène comprenant un ou plusieurs conjugués saccharides capsulaires de streptococcus pneumoniae et un constituant protéique comprenant la protéine e et/ou pila d'haemophilus influenzae

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JP6236086B2 (ja) 2017-11-22
WO2014060383A1 (fr) 2014-04-24
MX365842B (es) 2019-06-17
MX2015004949A (es) 2015-07-06
CN104853768B (zh) 2019-04-19
BR112015008419A8 (pt) 2019-09-10
CN104853768A (zh) 2015-08-19
BR112015008417A2 (pt) 2017-07-04
BR112015008419A2 (pt) 2017-07-04
EP2908856B1 (fr) 2019-02-20
IL238053A0 (en) 2015-05-31
CA2888310A1 (fr) 2014-04-24
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CA2888321A1 (fr) 2014-04-24
BR112015008419B1 (pt) 2022-11-08
AU2013331781A8 (en) 2015-05-14
ES2640320T3 (es) 2017-11-02
JP2015536312A (ja) 2015-12-21
WO2014060385A1 (fr) 2014-04-24
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CN104968366A (zh) 2015-10-07
EP2908855A1 (fr) 2015-08-26
IL238079A0 (en) 2015-05-31
EA201590490A1 (ru) 2015-10-30
WO2014060389A2 (fr) 2014-04-24
EP2908855B1 (fr) 2017-06-28
WO2014060389A3 (fr) 2014-09-12
AU2013333975A1 (en) 2015-05-07
EP2908856A1 (fr) 2015-08-26
MX2015005002A (es) 2015-07-17
BR112015008417A8 (pt) 2019-09-10
BR112015008418A2 (pt) 2017-07-04
AU2013331781A1 (en) 2015-05-07
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SG11201502635SA (en) 2015-05-28
CA2888300A1 (fr) 2014-04-24

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