HK1242601B - Immunogenic compositions for use in pneumococcal vaccines - Google Patents

Description

Immunogenic compositions for use in pneumococcal vaccines

Field of the Invention

The present invention relates to novel immunogenic compositions for use in pneumococcal vaccines. The immunogenic compositions of the invention typically comprise conjugated capsular saccharide antigens (glycoconjugates), wherein the saccharide is derived from a serotype of Streptococcus pneumoniae. One object of the present invention is to provide immunogenic compositions for protection against S. pneumoniae serogroup 9.

Background of the Invention

Infections caused by Streptococcus pneumoniae are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia, and meningitis are the most common manifestations of invasive pneumococcal disease, while dissemination of the bacteria within the respiratory tract can lead to middle ear infections, sinusitis, or recurrent bronchitis. Non-invasive manifestations are generally less severe than invasive disease but are considered more common.

Streptococcus pneumoniae, the causative agent of pneumococcal disease, is a Gram-positive, encapsulated coccus surrounded by a polysaccharide capsule. Variation in the composition of this capsule allows for serotype differentiation among approximately 91 capsular types, some of which are commonly associated with pneumococcal disease, while others are rare. Invasive pneumococcal infections include pneumonia, meningitis, and febrile bacteremia; common noninvasive manifestations are otitis media, sinusitis, and bronchitis.

Pneumococcal conjugate vaccine (PCV) is a pneumococcal vaccine used to protect against diseases caused by Streptococcus pneumoniae. Currently, three PCV vaccines are available on the global market: (known as Prevenar in some countries) (seven-valent vaccine), (decavalent vaccine), and (thirteen-valent vaccine).

The specific serotypes causing disease out of the 13 serotypes vary by region, population, and may change over time due to acquisition of antibiotic resistance, introduction of pneumococcal vaccines, and secular trends of unknown origin.

Incorporation of conjugates into immunogenic compositions is not a simple process, as combining conjugates into a single multivalent injection can result in competition between the different components and can adversely affect the immunogenicity of any individual conjugate.

This interference phenomenon can limit the number of conjugates that can be included in a multivalent vaccine. Thus, protecting against a large number of serotypes while limiting the number of conjugates in a composition, while of great value, may be difficult to achieve.

It is an object of the present invention to provide an immunogenic composition that adequately protects against S. pneumoniae, in particular S. pneumoniae serotype 9, while limiting the number of conjugates.

S. pneumoniae serotype 9 includes four different types: 9V, 9A, 9L and 9N, each of which produces its own type-specific capsular polysaccharide (Richards, J.C and M.B. Perry, 1988, In A.M. Wu (ed.), The molecular immunology complex carbohydrates. Plenum, New York, pp. 593-594).

It is an object of the present invention to provide immunogenic compositions with a limited number of conjugates that adequately protect against S. pneumoniae serotypes 9V, 9A, 9L and 9N.

SUMMARY OF THE INVENTION

The present invention relates to an immunogenic composition comprising at least one saccharide conjugate from S. pneumoniae serotype 9V for use in a method of immunizing a subject against infection by S. pneumoniae serotypes 9N, 9A and/or 9L. Preferably, the composition does not comprise capsular saccharides from S. pneumoniae serotypes 9N, 9A and 9L.

One aspect of the present invention relates to the use of an immunogenic composition comprising at least one saccharide conjugate from S. pneumoniae serotype 9V for the preparation of a medicament for immunizing a subject against infection by S. pneumoniae serotypes 9N, 9A and/or 9L. Preferably, the composition does not comprise capsular saccharides from S. pneumoniae serotypes 9N, 9A and 9L.

In one aspect, the immunogenic composition further comprises at least one saccharide conjugate from S. pneumoniae serotypes 4, 6B, 14, 18C, 19F and/or 23F.

In one aspect, the immunogenic composition further comprises at least one saccharide conjugate from S. pneumoniae serotype 1, 5 and/or 7F.

In one aspect, the immunogenic composition further comprises at least one saccharide conjugate from S. pneumoniae serotypes 6A and/or 19A.

In one aspect, the immunogenic composition further comprises at least one saccharide conjugate from serotypes 3, 15B, 22F, 33F, 12F, 10A, 11A and/or 8 of Streptococcus pneumoniae.

In yet another aspect, the immunogenic composition further comprises at least one saccharide conjugate from serotypes 2, 15C, 17F and/or 20 of Streptococcus pneumoniae.

In a further aspect, the immunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 valent pneumococcal conjugate composition.

In yet another aspect, the glycoconjugate of the immunogenic composition is conjugated solely to CRM197.

In one aspect, the saccharide conjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of the immunogenic composition are individually conjugated to PD, and, if present, the saccharide conjugate from S. pneumoniae serotype 18C is conjugated to TT and the saccharide conjugate from S. pneumoniae serotype 19F is conjugated to DT.

In one aspect, the glycoconjugate is prepared using CDAP chemistry or by reductive amination chemistry.

The immunogenic composition may further comprise antigens from other pathogens, and/or at least one adjuvant, such as aluminum phosphate, aluminum sulfate or aluminum hydroxide.

In one aspect, the immunogenic composition can elicit IgG antibodies in vivo at a concentration of at least 0.35 μg/ml as tested by ELISA, wherein the IgG antibodies can bind to S. pneumoniae serotype 9N, 9A and/or 9L polysaccharides.

In one aspect, the immunogenic composition is capable of eliciting a titer of at least 1 :8 against S. pneumoniae serotypes 9N, 9A, and/or 9L in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay (OPA).

In one aspect, the immunogenic composition significantly increases the proportion of responders to S. pneumoniae serotypes 9N, 9A and/or 9L compared to a previously immunized population.

In one aspect, the immunogenic composition significantly increases OPA titers against S. pneumoniae serotypes 9N, 9L, and/or 9A in human subjects compared to a previously immunized population.

In one aspect, the immunogenic composition is for use in a method of immunizing a subject against infection by S. pneumoniae serotypes 9N, 9L, and/or 9A.

In one aspect, the immunogenic composition is administered systemically or by a mucosal route and is used in a method for preventing, treating or ameliorating an infection, disease or condition caused by S. pneumoniae serotype 9N, 9A and/or 9L in a subject, for preventing S. pneumoniae serotype 9N, 9A and/or 9L infection in a subject, or for protecting or treating a human susceptible to S. pneumoniae serotype 9N, 9A and/or 9L infection.

In one aspect, the present invention relates to the use of the immunogenic composition disclosed in the present application document in the preparation of a medicament for administering the immunogenic composition by systemic or mucosal routes to prevent, treat or alleviate an infection, disease or condition caused by Streptococcus pneumoniae serotypes 9N, 9A and/or 9L in a subject, to prevent Streptococcus pneumoniae serotypes 9N, 9A and/or 9L infection in a subject, or to use in a method of protecting or treating a population susceptible to Streptococcus pneumoniae serotypes 9N, 9A and/or 9L infection.

In one aspect, the present invention relates to a method for preventing, treating or ameliorating an infection, disease or condition associated with S. pneumoniae serotypes 9N, 9A and/or 9L in a subject, comprising administering to the subject an immunologically effective amount of the immunogenic composition of the present invention.

In one aspect, the invention relates to a method of preventing infection with S. pneumoniae serotype 9N, 9A and/or 9L in a subject, comprising administering to the subject an immunologically effective amount of the immunogenic composition of the invention.

The present invention further relates to a kit comprising the immunogenic composition disclosed herein and an information leaflet referring to the ability of the composition to elicit functional antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N and a procedure for producing the kit.

WO2013/191459 discloses a 15-valent immunogenic composition comprising saccharide conjugates from S. pneumoniae serotypes 9N and 9V. Similarly, WO2014/092377 and Chinese patent application numbers CN 101590224 and CN 103656631 disclose immunogenic compositions combining saccharide conjugates from S. pneumoniae serotypes 9N and 9V.

Surprisingly, it was found that the serotype 9V polysaccharide conjugate can stimulate functional cross-reactive antibodies to other serotypes in serogroup 9 (9A, 9L and/or 9N) in addition to stimulating functional reactive antibodies to serogroup 9V.

Attached photos

Figure 1: Cross-functional OPA responses. A subset of 59 sera from adults immunized with the 13-valent pneumococcal conjugate vaccine (US study 6115A1-004; ClinicalTrials.gov Identifier: NCT00427895) was evaluated for the presence of functional antibodies against serotypes 9V, 9A, 9L, and 9N in OPA. The percentage of samples with a positive OPA titer (i.e., ≥1:8) is shown above each group. The geometric mean titer (GMT) is listed on the x-axis below each group.

Figure 2: Cross-functional OPA response of 66 matched pre- and post-vaccination sera. A subset of 66 matched pre- and post-vaccination serum panels from adults immunized with a 13-valent pneumococcal conjugate vaccine (study project 6115A1-3005; ClinicalTrials.gov Identifier: NCT00546572) was evaluated for the presence of functional antibodies in OPA against serotypes 9V, 9A, 9L, and 9N. The percentage of samples with a positive OPA titer (i.e., ≥1:8) is shown above each group. The x-axis below each group lists the geometric mean titer (GMT).

Figure 3: Reverse cumulative distribution curve (RCDC) before and after immunization with pneumococcal serotype 9V (Pn9V).

Reverse cumulative distribution curve of OPA titers for serotype 9V, serotype 9V derived from matched pre- and post-vaccination sera cohorts (N=66) immunized with 13-valent pneumococcal conjugate vaccine (Study Project 6115A1-3005; ClinicalTrials.gov Identifier: NCT00546572). Plot points represent the percentage of sera with positive OPA titers (i.e., ≥1:8).

Figure 4: Reverse cumulative distribution curves (RCDC) before and after Pneumococcal serotype 9A (Pn9A) immunization.

Reverse cumulative distribution curve of OPA titers for serotype 9A, derived from matched pre- and post-vaccination sera cohorts (N=66) immunized with the 13-valent pneumococcal conjugate vaccine (Study 6115A1-3005; ClinicalTrials.gov Identifier: NCT00546572). The plot points represent the percentage of sera with a positive OPA titer (i.e., ≥1:8).

Figure 5: Reverse cumulative distribution curves (RCDC) before and after Pneumococcal serotype 9L (Pn9L) immunization.

Reverse cumulative distribution curve of OPA titers for serotype 9L, derived from matched pre- and post-vaccination sera cohorts (N=66) immunized with the 13-valent pneumococcal conjugate vaccine (Study 6115A1-3005; ClinicalTrials.gov Identifier: NCT00546572). The plot points represent the percentage of sera with a positive OPA titer (i.e., ≥1:8).

Figure 6: Reverse cumulative distribution curves (RCDC) before and after immunization with Pneumococcal serotype 9N (Pn9N).

Reverse cumulative distribution curve of OPA titers for serotype 9N, derived from matched pre- and post-vaccination sera cohorts (N=66) immunized with the 13-valent pneumococcal conjugate vaccine (Study 6115A1-3005; ClinicalTrials.gov Identifier: NCT00546572). The plot points represent the percentage of sera with a positive OPA titer (i.e., ≥1:8).

Figure 7: Serotype 9 cross-functional OPA responses of matched pre/post vaccination sera from study B1851088 (ClinicalTrials.gov Identifier: NCT01646398) following immunization with 13vPnC.

A subset of 91 matched pre- and post-vaccination serum cohorts from adults vaccinated with 13vPnC (research project B1851088) was evaluated for the presence of functional antibodies against serotypes 9V, 9A, 9L, and 9N in OPA. The percentage of samples with positive OPA titers (i.e., ≥1:8) is shown above each group.

Figure 8: Serotype 9 cross-functional OPA responses of matched pre/post vaccination sera from study B1851088 following immunization with 23 vPS.

A subset of 83 matched pre- and post-vaccination serum cohorts from adults vaccinated with 23vPS (research project B1851088) was evaluated for the presence of functional antibodies against serotypes 9V, 9A, 9L, and 9N in OPA. The percentage of samples with positive OPA titers (i.e., ≥1:8) is shown above each group.

Figure 9: Serotype 9 cross-functional OPA responses of matched pre/post vaccination sera from study B1851088 following immunization with 13vPnC (13v) or 23vPS (23v).

Figure 10: Reverse cumulative distribution curve (RCDC) of Pn9V & Pn9N 13vPnC from research project B1851088.

Reverse cumulative distribution curves of OPA titers for serotypes 9V and 9N derived from matched pre- and post-vaccination sera cohorts (N=91) vaccinated with 13vPnC (Japanese research project B1851088).

Figure 11: Reverse cumulative distribution curve (RCDC) of Pn9V & Pn9N 23vPS from research project B1851088.

Reverse cumulative distribution curves of OPA titers for serotypes 9V and 9N derived from matched pre- and post-vaccination sera cohorts (N=83) vaccinated with 23vPS (Japanese research project B1851088).

Figure 12: Reverse cumulative distribution curve (RCDC) of Pn9V & Pn9A 13vPnC from research project B1851088.

Reverse cumulative distribution curves of OPA titers for serotypes 9V and 9A derived from matched pre- and post-vaccination sera cohorts (N=91) vaccinated with 13vPnC (Japanese research project B1851088).

Figure 13: Reverse cumulative distribution curve (RCDC) of Pn9V & Pn9A 23vPS from research project B1851088.

Reverse cumulative distribution curves of OPA titers for serotypes 9V and 9A derived from matched pre- and post-vaccination sera cohorts (N=83) vaccinated with 23vPS (Japanese research project B1851088).

Figure 14: Pn9V & Pn9L 13vPnC reverse cumulative distribution curve (RCDC) from research project B1851088.

Reverse cumulative distribution curves of OPA titers for serotypes 9V and 9L derived from matched pre- and post-vaccination sera cohorts (N=91) vaccinated with 13vPnC (Japanese research project B1851088).

Figure 15: Reverse cumulative distribution curve (RCDC) of Pn9V & Pn9L 23vPS from research project B1851088.

Reverse cumulative distribution curves of OPA titers for serotypes 9V and 9L derived from matched pre- and post-vaccination sera cohorts (N=83) vaccinated with 23vPS (Japanese research project B1851088).

1. Immunogenic compositions of the present invention

The immunogenic compositions of the invention typically comprise conjugated capsular saccharide antigens (also known as glycoconjugates) wherein the saccharide is derived from a serotype of S. pneumoniae.

Preferably, the number of S. pneumoniae capsular saccharides can be from 1 serotype (or "v", valence) to 24 different serotypes (24v). In one embodiment, there is 1 serotype. In one embodiment, there are 2 different serotypes. In one embodiment, there are 3 different serotypes. In one embodiment, there are 4 different serotypes. In one embodiment, there are 5 different serotypes. In one embodiment, there are 6 different serotypes. In one embodiment, there are 7 different serotypes. In one embodiment, there are 8 different serotypes. In one embodiment, there are 9 different serotypes. In one embodiment, there are 10 different serotypes. In one embodiment, there are 11 different serotypes. In one embodiment, there are 12 different serotypes. In one embodiment, there are 13 different serotypes. In one embodiment, there are 14 different serotypes. In one embodiment, there are 15 different serotypes. In one embodiment, there are 16 different serotypes. In one embodiment, there are 17 different serotypes. In one embodiment, there are 18 different serotypes. In one embodiment, there are 19 different serotypes. In one embodiment, there are 20 different serotypes. In one embodiment, there are 21 different serotypes. In one embodiment, there are 22 different serotypes. In one embodiment, there are 23 different serotypes. In one embodiment, there are 24 different serotypes. As described below, the capsular saccharide is conjugated to a carrier protein to form a glycoconjugate.

If the protein carrier is identical for two or more saccharides in the composition, the saccharides can be conjugated to the same molecule of protein carrier (the carrier molecule having two or more different saccharides conjugated thereto) [see, for example, WO 2004/083251].

In a preferred embodiment, the saccharides are each individually conjugated to different molecules of a protein carrier (each molecule of protein carrier has only one type of saccharide conjugated thereto). In such embodiments, the capsular saccharides are considered to be individually conjugated to the carrier protein.

For the purposes of this invention, the term "glycoconjugate" refers to a capsular saccharide covalently linked to a carrier protein. In one embodiment, the capsular saccharide is directly linked to the carrier protein. In another embodiment, the bacterial saccharide is linked to the protein via a spacer/linker.

1.1 Carrier Protein of the Present Invention

One component of the glycoconjugates of the present invention is a carrier protein to which the glycoconjugate is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" are used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures.

In a preferred embodiment, the carrier protein of the glycoconjugate is selected from the group consisting of DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM ₁₉₇ (a non-toxic but antigenically identical variant of diphtheria toxin), diphtheria toxin A chain mutant CRM ₁₉₇ (CN103495161), other DT mutants (such as CRM176, CRM228, CRM45 (Uchida et al. (1973) J. Biol. Chem. 218: 3838-3844), CRM9, CRM102, CRM103 or CRM107; and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc. (1992); deletion of Glu-148 or mutation of Glu-148 to Asp, Gln or Ser, and/or Ala 158 to Gly, and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; mutations of at least one or more residues of Lys 516, Lys 526, Phe 530 and/or Lys 534, and other mutations disclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or fragments disclosed in U.S. Pat. No. 5,843,711; pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect Immun 63:2706-2713), including detoxified forms of ply, such as dPLY-GMBS (WO 2004/081515 and WO 2006/032499) or dPLY-formol, PhtX (including PhtA, PhtB, PhtD, PhtE (the sequence of PhtA, PhtB, PhtD or PhtE is disclosed in WO 00/37105 and WO 00/39299)) and fusion proteins of Pht proteins (e.g., PhtDE fusion protein, PhtBE fusion protein, Pht AE (WO 01/98334, WO 03/054007, WO 2009/000826)), OMPC (meningococcal outer membrane protein - usually extracted from Neisseria meningitidis serotype B (EP0372501)), PorB (from Neisseria meningitidis), PD (from Haemophilus influenzae influenzae) protein D; see, for example, EP0594610 B), or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins comprising multiple human CD4+ T cell epitopes from antigens derived from various pathogens (Falugi et al. (2001) Eur J Immunol 31:3816-3824) such as N19 protein (Baraldoi et al. (2004) Infect lmmun 72:4884-4887), pneumococcal surface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337), Clostridium difficile toxin A or B (WO 00/61761), transferrin binding protein, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (particularly non-toxic mutants thereof (e.g., exotoxin A having a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971)). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or a purified protein derivative of tuberculin (PPD) can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g., as described in WO 2004/083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.

In a preferred embodiment, the carrier protein of the glycoconjugate is individually selected from the group consisting of TT, DT, DT mutants (such as _CRM197 ), Haemophilus influenzae protein D, PhtX, PhtD, PhtDE fusion proteins (particularly those described in WO 01/98334 and WO 03/054007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B of Clostridium difficile and PsaA.

In one embodiment, the carrier protein of the glycoconjugate of the present invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the glycoconjugate of the present invention is TT (tetanus toxoid).

In another embodiment, the carrier protein of the glycoconjugate of the invention is PD (Haemophilus influenzae protein D; see, eg, EP 0 594 610 B).

The CRM ₁₉₇ protein is a non-toxic form of diphtheria toxin, but it is immunologically indistinguishable from diphtheria toxin. CRM ₁₉₇ is produced by Corynebacterium diphtheriae infected with the non-toxin-producing phage β197 ^tox- , which is produced by nitrosoguanidine mutagenesis of the toxin-producing β-corynebacterial phage (Uchida et al. (1971) Nature New Biology 233:8-11). The CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin, but differs from it by a single base change in the structural gene (guanine is changed to adenine). This single base change results in an amino acid substitution in the mature protein (glutamic acid instead of glycine) and eliminates the toxic properties of diphtheria toxin. The CRM ₁₉₇ protein is a safe and effective T cell-dependent carrier of sugars. Further detailed descriptions of CRM ₁₉₇ and its production can be found in, for example, U.S. Patent No. 5,614,382. In one embodiment, the capsular saccharide of the present invention is conjugated to the CRM ₁₉₇ protein or the A chain of CRM ₁₉₇ (see CN103495161). In one embodiment, the capsular saccharide of the present invention is conjugated to the A chain of CRM ₁₉₇ obtained by genetic recombinant Escherichia coli expression (see CN103495161). In one embodiment, the capsular saccharide of the present invention is conjugated to CRM _197. In one embodiment, the capsular saccharide of the present invention is conjugated to the A chain of CRM ₁₉₇ .

Thus, in a common embodiment, the glycoconjugates of the invention comprise CRM ₁₉₇ as a carrier protein, wherein the capsular polysaccharide is covalently linked to CRM ₁₉₇ .

1.2 Capsular saccharides of the present invention

In this specification, the term "saccharide" may refer to a polysaccharide or an oligosaccharide and includes both. In a common embodiment, the saccharide is a polysaccharide, in particular a Streptococcus pneumoniae capsular polysaccharide.

Capsular polysaccharides are prepared by standard techniques known to those skilled in the art.

In the present invention, capsular polysaccharides can be prepared, for example, from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F of S. pneumoniae. Typically, capsular polysaccharides are produced by growing each serotype of S. pneumoniae in a culture medium (e.g., a soy-based culture medium) and then preparing the polysaccharide from the bacterial culture. The S. pneumoniae bacterial strain used to produce the individual polysaccharides used in the saccharide conjugates of the present invention can be obtained from established culture collections or clinical samples.

Typically, the population of each S. pneumoniae serotype is scaled up from a seed vial to a seed culture bottle and increased in volume through one or more seed fermentors until the production-scale fermentation volume is reached. At the end of the growth cycle, the cells are lysed and the lysate broth is harvested for downstream (purification) processing (see, for example, WO 2006/110381, WO 2008/118752, and U.S. Patent Application Publication Nos. 2006/0228380, 2006/0228381, 2008/0102498, and 2008/0286838).

Individual polysaccharides are typically purified by centrifugation, precipitation, ultrafiltration and/or column chromatography (see, for example, WO 2006/110352 and WO 2008/118752).

As further described herein, the purified polysaccharide may be rendered active (eg, chemically activated) to enable it to react (eg, directly with a carrier protein or through a linker such as an eTEC spacer) and subsequently incorporated into the glycoconjugates of the invention.

The S. pneumoniae capsular polysaccharide comprises repeating oligosaccharide units that may contain up to 8 sugar residues.

In one embodiment, the capsular saccharide of the invention may be an oligosaccharide unit, or a sugar chain shorter than the natural length of repeating oligosaccharide units. In one embodiment, the capsular saccharide of the invention is a repeating oligosaccharide unit of the relevant serotype.

In one embodiment, the capsular saccharide of the invention may be an oligosaccharide. Oligosaccharides have a relatively small number of repeating units (typically 5-15 repeating units) and are typically derived synthetically or by hydrolysis of polysaccharides.

Preferably, however, all capsular saccharides of the present invention and in the immunogenic compositions of the present invention are polysaccharides. High molecular weight capsular polysaccharides are capable of inducing an immune response with certain antibodies due to the presence of epitopes on their antigenic surfaces. Isolation and purification of high molecular weight capsular polysaccharides are preferably contemplated for use in the conjugates, compositions, and methods of the present invention.

In some embodiments, the polysaccharide has a molecular weight of 5 kDa to 4,000 kDa prior to conjugation. In other such embodiments, the polysaccharide has a molecular weight of 10 kDa to 4,000 kDa, 50 kDa to 4,000 kDa, 50 kDa to 3,000 kDa, 50 kDa to 2,000 kDa, 50 kDa to 1,500 kDa, 50 kDa to 1,000 kDa, 50 kDa to 750 kDa, 50 kDa to 500 kDa, 100 kDa to 4,000 kDa, 100 kDa to 3,0 ...2,000 kDa, 50 kDa to 1,500 kDa, 50 kDa to 1,000 kDa, 50 kDa to 750 kDa, 50 kDa to 500 kDa, 100 kDa to 4,000 kDa, 100 kDa to 3,000 kDa, 100 kDa to 2,000 kDa, 2,000kDa, 100kDa-1,500kDa, 100kDa-1,000kDa, 100kDa-750kDa, 100kDa-500kDa, 100-400kDa, 200kDa- 4,000kDa, 200kDa-3,000kDa, 200kDa-2,000kDa, 200kDa-1,500kDa, 200kDa-1,000kDa or 200kDa-500kDa.

In further embodiments, the capsular polysaccharide has a molecular weight of 70 kDa-150 kDa, 80 kDa-160 kDa, 90 kDa-250 kDa, 100 kDa-1,000 kDa, 100 kDa-500 kDa, 100 kDa-400 kDa, 100 kDa-160 kDa, 150 kDa-600 kDa, 200 kDa-1,000 kDa, 200 kDa-600 kDa, 200 kDa-400 kDa, 300 kDa-1,000 kDa, 300 kDa-600 kDa, 300 kDa-500 kDa, or 500 kDa-600 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

The size of the polysaccharide may be slightly reduced during normal purification procedures. In addition, as described herein, the polysaccharide may be subjected to sizing techniques prior to conjugation. Mechanical or chemical sizing may be applied. Chemical hydrolysis may be performed using acetic acid. Mechanical sizing may be performed using high pressure homogenization shearing. The molecular weight ranges mentioned above refer to the purified polysaccharide prior to conjugation (e.g., prior to activation).

In a preferred embodiment, the purified polysaccharide is a capsular polysaccharide from S. pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F, wherein the molecular weight of the capsular polysaccharide falls within one of the above molecular weight ranges.

As used herein, the term "molecular weight" of a polysaccharide or carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) combined with multi-angle laser light scattering detection (MALLS).

In some embodiments, the pneumococcal saccharides of the invention from serotypes 9V, 18C, 11A, 15B, 22F and/or 33F are O-acetylated. In some embodiments, the pneumococcal saccharides of the invention from serotypes 9V, 11A, 15B, 22F and/or 33F are O-acetylated.

The degree of O-acetylation of the polysaccharide can be determined by any method known in the art, for example by proton NMR (see, for example, Lemercinier et al. (1996) Carbohydrate Research 296:83-96, Jones et al. (2002) J. Pharmaceutical and Biomedical Analysis 30:1233-1247, WO 2005/033148 and WO 00/56357). Another commonly used method is described in Hestrin (1949) J. Biol. Chem. 180:249-261. Preferably, the presence of O-acetyl groups is determined by ion-HPLC analysis.

The purified polysaccharides described herein are chemically activated to produce saccharides that are reactive with carrier proteins. As described below, these pneumococcal conjugates are prepared by separate procedures and formulated as single dose formulations.

1.3 Glycoconjugates of the Invention

The purified saccharide is chemically activated to produce a saccharide that can react with a carrier protein directly or through a linker (i.e., an activated saccharide). Once activated, each capsular saccharide is conjugated to a carrier protein to form a glycoconjugate. In one embodiment, each capsular saccharide is conjugated to the same carrier protein. The chemical activation of the saccharide and subsequent conjugation to the carrier protein can be achieved by the activation and conjugation methods disclosed herein.

Capsular polysaccharides from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F are prepared as described above.

In one embodiment, the polysaccharide is activated with 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then conjugated to an amino group on a carrier protein (preferably CRM ₁₉₇ ) directly or through a spacer (linker) group. For example, the spacer can be cystamine or cysteamine to provide a thiolated polysaccharide, which can be bound to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy] succinimide ester (GMBS)) or a halogenated carrier protein (e.g., using iodoacetamide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SlAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamide] propionate (SBAP)). Preferably, the cyanate ester (optionally generated by CDAP chemistry) is combined with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized sugar is conjugated to a carrier protein (e.g., CRM ₁₉₇ ) via the carboxyl groups on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugations are described, for example, in WO 93/15760, WO 95/08348, and WO 96/129094.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F are prepared by using CDAP chemistry. In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F and 23F are prepared by using CDAP chemistry. In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 14, 18C, 19F and 23F are prepared by using CDAP chemistry. In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19A, 19F and 23F are prepared by using CDAP chemistry. In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 14, 18C, 19A, 19F and 23F are prepared by using CDAP chemistry. In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 14, 18C, 19A, 19F and 23F are prepared by using CDAP chemistry.

Other suitable conjugation techniques utilize carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in International Patent Application Publication No. WO 98/42721. Conjugation can involve a carbonyl linker, which can be formed by reacting a free hydroxyl group of a sugar with CDI (see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), followed by reaction with the protein to form a carbamate bond. This can include reduction of the anomeric end 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 conjugation of the CDI carbamate intermediate to an amino group on the protein.

In a preferred embodiment, at least one capsular polysaccharide from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F is conjugated to a carrier protein by a reductive amination reaction (as described in U.S. Patent Application Publication Nos. 2006/0228380, 2007/184072, 2007/0231340, and 2007/0184071, WO 2006/110381, WO 2008/079653, and WO 2008/143709).

In one embodiment of the present invention, the glycoconjugate from Streptococcus pneumoniae serotype 6A is prepared by a reductive amination reaction. In one embodiment of the present invention, the glycoconjugate from Streptococcus pneumoniae serotype 19A is prepared by a reductive amination reaction. In one embodiment of the present invention, the glycoconjugate from Streptococcus pneumoniae serotype 3 is prepared by a reductive amination reaction. In one embodiment of the present invention, the glycoconjugate from Streptococcus pneumoniae serotypes 6A and 19A is prepared by a reductive amination reaction. In one embodiment of the present invention, the glycoconjugate from Streptococcus pneumoniae serotypes 3, 6A and 19A is prepared by a reductive amination reaction.

In a preferred embodiment of the present invention, the glycoconjugates from Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F and 23F are prepared by reductive amination. In one embodiment of the present invention, the glycoconjugates from Streptococcus pneumoniae serotypes 1, 4, 6B, 9V, 14, 18C, 19F and 23F are prepared by reductive amination. In one embodiment of the present invention, the glycoconjugates from Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F are prepared by reductive amination. In one embodiment of the present invention, the glycoconjugates from Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F are prepared by reductive amination. In one embodiment of the invention, the glycoconjugates from Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F are prepared by reductive amination. In one embodiment of the invention, the glycoconjugates from Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are prepared by reductive amination. In one embodiment of the invention, the glycoconjugates from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F and 23F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F and 23F are prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are all prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15C, 18C, 19A, 19F, 22F, 23F and 33F are prepared by reductive amination.

In another preferred embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F are prepared by reductive amination.

The reductive amination reaction involves two steps: (1) oxidation of the polysaccharide, and (2) reduction of the activated polysaccharide with the carrier protein to form a conjugate. Prior to oxidation, the polysaccharide is optionally hydrolyzed. Mechanical or chemical hydrolysis methods can be used. Chemical hydrolysis methods can be performed using acetic acid.

The oxidation step may include reaction with periodate. For the purposes of the present invention, the term "periodate" includes periodate and periodic acid; the term also includes metaperiodate (IO ₄ ⁻ ) and orthoperiodate (IO ₆ ^{5 −} ), as well as various salts of periodic acid (e.g., sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably sodium periodate (NaIO ₄ ). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably periodic acid.

In one embodiment, the oxidant is a stable nitroxyl or nitroxide radical compound, such as a piperidine-N-oxy or pyrrolidine-N-like compound, which selectively oxidizes primary hydroxyl groups in the presence of an oxidant (as described in WO 2014/097099). In the reaction, the actual oxidant in the catalytic cycle is an N-oxoammonium salt. In one aspect, the stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one aspect, the stable nitroxyl or nitroxide radical compound has a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, the stable nitroxide radical compound is TEMPO or a derivative thereof. In one aspect, the oxidant is a molecule having an N-halogen moiety. In one aspect, the oxidant is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-thiazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-thiazinane-2,4,6-trione, diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-thiazinane-2,4,6-trione. Preferably, the oxidant is N-chlorosuccinimide.

In a preferred embodiment, the capsular polysaccharide from Streptococcus pneumoniae serotype 12F is conjugated to a carrier protein via a reductive amination reaction, wherein the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) is used as a co-oxidizing agent (as described in WO 2014/097099). Thus, in one aspect, a saccharide conjugate from Streptococcus pneumoniae serotype 12F can be obtained by a method comprising the following steps: a) reacting the 12F saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amine groups (hereinafter referred to as "TEMPO/NCS reductive amination reaction").

Optionally, the oxidation reaction is quenched by adding a quencher. The quencher can be selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfites, bisulfates, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphites or (such as glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid).

After the polysaccharide oxidation step, the polysaccharide is said to be activated, hereinafter referred to as "activated polysaccharide". The activated polysaccharide and carrier protein can be lyophilized (freeze-dried) separately (independent lyophilization) or together (co-lyophilization). In one embodiment, the activated polysaccharide and carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and carrier protein are lyophilized separately.

In one embodiment, lyophilization occurs in the presence of a non-reducing sugar, possible non-reducing sugars including sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinitol.

The second step of the conjugation procedure is to reduce the activated polysaccharide and the carrier protein using a reducing agent to form a conjugate (so-called reductive amination reaction). Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride, sodium triacetoxyborohydride, or sodium or zinc borohydride in the presence of Bronsted or Lewis acid), amine boranes such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine borane, t-BuMe ⁱ PrN-BH ₃ , benzylamine-BH ₃ or 5-ethyl-2-picoline borane (PEMB) or borohydride exchange resins. In one embodiment, the reducing agent is sodium cyanoborohydride.

In one embodiment, the reduction reaction is carried out in an aqueous solvent (e.g., selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine or HEPB, pH 6.0-8.5, 7.0-8.0 or 7.0-7.5). In another embodiment, the reaction is carried out in an aprotic solvent. In one embodiment, the reduction reaction is carried out in DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. DMSO or DMF solvent can be used to reconstitute activated polysaccharides and carrier proteins that have been freeze-dried.

At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, this capping agent is sodium borohydride (NaBH ₄ ). After conjugation (reduction reaction and optional capping), the glycoconjugate can be purified (enriched for the amount of polysaccharide-protein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration/diafiltration procedures, tangential flow filtration precipitation/elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. In one embodiment, the glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

In one embodiment, the glycoconjugate is sterile filtered.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F and 23F are prepared using CDAP chemistry and the glycoconjugate from S. pneumoniae serotype 6A is prepared by reductive amination.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F and 23F are prepared using CDAP chemistry and the glycoconjugate from S. pneumoniae serotype 19A is prepared by reductive amination.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F and 23F are prepared using CDAP chemistry and the glycoconjugates from S. pneumoniae serotypes 6A and 19A are prepared by reductive amination.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F and 23F are prepared using CDAP chemistry and the glycoconjugates from S. pneumoniae serotypes 3, 6A and 19A are prepared by reductive amination.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F and 23F are prepared using CDAP chemistry and the glycoconjugate from S. pneumoniae serotype 6A is prepared by reductive amination.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F and 23F are prepared using CDAP chemistry and the glycoconjugate from S. pneumoniae serotype 19A is prepared by reductive amination.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F and 23F are prepared using CDAP chemistry and the glycoconjugates from S. pneumoniae serotypes 6A and 19A are prepared by reductive amination.

In one embodiment of the invention, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 8, 9V, 14, 18C, 19F, 22F and 23F are prepared using CDAP chemistry and the glycoconjugates from S. pneumoniae serotypes 3, 6A and 19A are prepared by reductive amination.

In one embodiment, the glycoconjugates of the present invention are prepared using an eTEC conjugation method as described in WO2014/027302. The glycoconjugates comprise a saccharide covalently conjugated to a carrier protein via one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer via a carbamate bond, and wherein the carrier protein is covalently conjugated to the eTEC spacer via an amide bond. The eTEC-linked glycoconjugates of the present invention can be represented by the general formula (I):

The atoms comprising the eTEC spacer are shown in the center box.

The eTEC spacer comprises seven linear atoms (i.e., –C(O)NH(CH ₂ ) ₂ SCH ₂ C(O)-) and provides stable thioether and amide bonds between the saccharide and the carrier protein. The synthesis of eTEC-linked glycoconjugates involves the reaction of an activated hydroxyl group of the saccharide with an amino group of a thioalkylamine reactant (e.g., cystamine or cysteamine or a salt thereof) to form a carbamate bond to the saccharide to provide a thiolated saccharide. One or more free sulfhydryl groups are generated by reaction with a reducing agent to provide an activated thiolated saccharide. The free sulfhydryl groups of the activated thiolated saccharide react with an activated carrier protein having one or more α-haloacetamide groups on an amine-containing residue to generate a thioether bond to form a conjugate, wherein the carrier protein is linked to the eTEC spacer via an amide bond.

In the saccharide conjugates of the present invention, the saccharide can be a polysaccharide or an oligosaccharide. The carrier protein can be selected from any suitable carrier as described herein or known to those skilled in the art. In conventional embodiments, the saccharide is a polysaccharide. In some such embodiments, the carrier protein is CRM _197. In some such embodiments, the eTEC-linked saccharide conjugate comprises a capsular polysaccharide of Streptococcus pneumoniae serotype 33F.

In a particularly preferred embodiment, the eTEC-linked glycoconjugate comprises pneumococcal serotype 33F (Pn33F) capsular polysaccharide covalently conjugated to CRM ₁₉₇ (serotype 33F eTEC-linked glycoconjugate) via an eTEC spacer.

In some embodiments, the glycoconjugates of the invention from S. pneumoniae serotypes 1, 7F, 9V, and/or 18C are O-acetylated. In some embodiments, the glycoconjugates from S. pneumoniae serotypes 1, 7F, and 9V are O-acetylated, and the glycoconjugate from S. pneumoniae serotype 18C is de-O-acetylated.

In some embodiments, the saccharide conjugate from Streptococcus pneumoniae serotype 1 comprises a saccharide having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%.

In some embodiments, the saccharide conjugate from Streptococcus pneumoniae serotype 7F comprises a saccharide having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%.

In some embodiments, the saccharide conjugate from S. pneumoniae serotype 9V comprises a saccharide having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%.

In some embodiments, the glycoconjugates from S. pneumoniae serotype 18C comprise saccharides having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is ≥10%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or about 100%. However, preferably, the glycoconjugates from S. pneumoniae serotype 18C are de-O-acetylated. In some of the embodiments, the saccharide conjugate from Streptococcus pneumoniae serotype 18C comprises saccharides with O-acetylation as follows: 0-50%, 0-40%, 0-30%, 0-20%, 0-10%, 0-5%, or 0-2%. In other embodiments, the degree of O-acetylation is ≤50%, ≤40%, ≤30%, ≤20%, ≤10%, ≤5%, ≤2%, or ≤1%.

Percent O-acetylation % refers to the percentage of a given sugar relative to 100% where each repeat unit is fully acetylated with respect to its acetylated structure.

In some embodiments, the glycoconjugates of the present invention comprise a saccharide having a molecular weight of 10 kDa to 2,000 kDa. In other such embodiments, the saccharide has a molecular weight of 50 kDa to 1,000 kDa. In other such embodiments, the saccharide has a molecular weight of 70 kDa to 900 kDa. In other such embodiments, the saccharide has a molecular weight of 100 kDa to 800 kDa. In other such embodiments, the saccharide has a molecular weight of 200 kDa to 600 kDa. In further such embodiments, the saccharide has a molecular weight of 100 kDa-1000 kDa, 100 kDa-900 kDa, 100 kDa-800 kDa, 100 kDa-700 kDa, 100 kDa-600 kDa, 100 kDa-500 kDa, 100 kDa-400 kDa, 100 kDa-300 kDa, 150 kDa-1,000 kDa, 150 kDa-900 kDa, 150kDa-800kDa, 150kDa-700kDa, 150kDa-600kDa, 150kDa-500kDa, 150kDa-400kDa, 150kDa-300kD a. 200kDa-1,000kDa, 200kDa-900kDa, 200kDa-800kDa, 200kDa-700kDa, 200kDa-600kDa, 200kDa-50 0kDa, 200kDa-400kDa, 200kDa-300, 250kDa-1,000kDa, 250kDa-900kDa, 250kDa-800kDa, 250kDa-7 00kDa, 250kDa-600kDa, 250kDa-500kDa, 250kDa-400kDa, 250kDa-350kDa, 300kDa-1,000kDa, 300kD kDa, 300kDa-900kDa, 300kDa-800kDa, 300kDa-700kDa, 300kDa-600kDa, 300kDa-500kDa, 300kDa-400kDa, 400kDa-1,000kDa, 400kDa-900kDa, 400kDa-800kDa, 400kDa-700kDa, 400kDa-600kDa, 500kDa-600kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure. In some such embodiments, the glycoconjugate is prepared using a reductive amination reaction.

In some embodiments, the molecular weight of the glycoconjugates of the present invention is 400 kDa-15,000 kDa, 500 kDa-10,000 kDa, 2,000 kDa-10,000 kDa, 3,000 kDa-8,000 kDa, or 3,000 kDa-5,000 kDa. In other embodiments, the molecular weight of the glycoconjugates is 500 kDa-10,000 kDa. In other embodiments, the molecular weight of the glycoconjugates is 1,000 kDa-8,000 kDa. In still other embodiments, the molecular weight of the glycoconjugates is 2,000 kDa-8,000 kDa or 3,000 kDa-7,000 kDa. In further embodiments, the molecular weight of the glycoconjugate of the present invention is 200 kDa-20,000 kDa, 200 kDa-15,000 kDa, 200 kDa-10,000 kDa, 200 kDa-7,500 kDa, 200 kDa-5,000 kDa, 200 kDa-3,000 kDa, 200 kDa-1,000 kDa, 500 kDa-20,000 kDa, 500 kDa-15,000 kDa, 500 kDa-12,500 kDa, 500 kDa-10,000 kDa, 00kDa, 500kDa-7,500kDa, 500kDa-6,000kDa, 500kDa-5,000kDa, 500kDa-4,000kDa, 500kDa-3,000kDa, 500kDa-2,000kDa , 500kDa-1,500kDa, 500kDa-1,000kDa, 750kDa-20,000kDa, 750kDa-15,000kDa, 750kDa-12,500kDa, 750kDa-10,000kDa, 7 50kDa-7,500kDa, 750kDa-6,000kDa, 750kDa-5,000kDa, 750kDa-4,000kDa, 750kDa-3,000kDa, 750kDa-2,000kDa, 750kDa -1,500kDa, 1,000kDa-15,000kDa, 1,000kDa-12,500kDa, 1,000kDa-10,000kDa, 1,000kDa-7,500kDa, 1,000kDa-6,000kDa kDa, 1,000 kDa-5,000 kDa, 1,000 kDa-4,000 kDa, 1,000 kDa-2,500 kDa, 2,000 kDa-15,000 kDa, 2,000 kDa-12,500 kDa, 2,000 kDa-10,000 kDa, 2,000 kDa-7,500 kDa, 2,000 kDa-6,000 kDa, 2,000 kDa-5,000 kDa, 2,000 kDa-4,000 kDa, or 2,000 kDa-3,000 kDa.

In further embodiments, the glycoconjugate of the invention has a molecular weight of 3,000 kDa-20,000 kDa, 3,000 kDa-15,000 kDa, 3,000 kDa-10,000 kDa, 3,000 kDa-7,500 kDa, 3,000 kDa-5,000 kDa, 4,000 kDa-20,000 kDa, 4,000 kDa-15,000 kDa, 4,000 kDa-12,500 kDa, 4,000 kDa-10,000 kDa, 4,000 kDa-7,500 kDa, 4,000 kDa-6,000 kDa, or 4,000 kDa-5,000 kDa.

In further embodiments, the glycoconjugate of the invention has a molecular weight of 5,000 kDa-20,000 kDa, 5,000 kDa-15,000 kDa, 5,000 kDa-10,000 kDa, 5,000 kDa-7,500 kDa, 6,000 kDa-20,000 kDa, 6,000 kDa-15,000 kDa, 6,000 kDa-12,500 kDa, 6,000 kDa-10,000 kDa, or 6,000 kDa-7,500 kDa.

The molecular weight of the glycoconjugate is measured by SEC-MALLS.Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

In a preferred embodiment, the serotype 22F saccharide conjugates of the present invention comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7, or about 0.8 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the saccharide conjugates comprise at least 0.5, 0.6, or 0.7 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the saccharide conjugates comprise at least 0.6 mM acetate per mM serotype 22F polysaccharide. In a preferred embodiment, the saccharide conjugates comprise at least 0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the serotype 33F saccharide conjugate of the present invention comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mM acetate/mM serotype 33F capsular polysaccharide. In a preferred embodiment, the saccharide conjugate comprises at least 0.5, 0.6 or 0.7 mM acetate/mM serotype 33F capsular polysaccharide. In a preferred embodiment, the saccharide conjugate comprises at least 0.6 mM acetate/mM serotype 33F capsular polysaccharide. In a preferred embodiment, the saccharide conjugate comprises at least 0.7 mM acetate/mM serotype 33F capsular polysaccharide. In a preferred embodiment, the presence of O-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the serotype 15B saccharide conjugates of the present invention comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mM acetate per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the saccharide conjugates comprise at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the saccharide conjugates comprise at least 0.6 mM acetate per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the saccharide conjugates comprise at least 0.7 mM acetate per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the presence of O-acetyl groups is determined by ion HPLC analysis.

In a preferred embodiment, the serotype 15B saccharide conjugates of the present invention comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mM glycerol per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the serotype 15B saccharide conjugates of the present invention comprise at least 0.5, 0.6 or 0.7 mM glycerol per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the serotype 15B saccharide conjugates of the present invention comprise at least 0.6 mM glycerol per mM serotype 15B capsular polysaccharide. In a preferred embodiment, the serotype 15B saccharide conjugates of the present invention comprise at least 0.7 mM glycerol per mM serotype 15B capsular polysaccharide.

In a preferred embodiment, the serotype 11A saccharide conjugate of the present invention comprises at least 0.3, 0.5, 0.6, 1.0, 1.4, 1.8, 2.2, 2.6, 3.0, 3.4, 3.8, 4.2, 4.6 or about 5.0 mM acetate per mM serotype 11A polysaccharide. In a preferred embodiment, the serotype 11A saccharide conjugate of the present invention comprises at least 1.8, 2.2 or 2.6 mM acetate per mM serotype 11A polysaccharide. In one embodiment, the saccharide conjugate comprises at least 0.6 mM acetate per mM serotype 11A polysaccharide. In a preferred embodiment, the serotype 11A saccharide conjugates of the present invention comprise at least 0.6, 1.0, 1.4, 1.8, 2.2, 2.6, 3.0, 3.4, 3.8, 4.2 or about 4.6 mM acetate/mM serotype 11A polysaccharide and less than about 5.0 mM acetate/mM serotype 11A polysaccharide. In one embodiment, the serotype 11A saccharide conjugates of the present invention comprise at least 0.6, 1.0, 1.4, 1.8, 2.2, 2.6 or about 3.0 mM acetate/mM serotype 11A polysaccharide and less than about 3.4 mM acetate/mM serotype 11A polysaccharide. In one embodiment, the serotype 11A saccharide conjugates of the present invention comprise at least 0.6, 1.0, 1.4, 1.8, 2.2, 2.6 or about 3.0 mM acetate/mM serotype 11A polysaccharide and less than about 3.3 mM acetate/mM serotype 11A polysaccharide. Any of the foregoing numbers are contemplated as embodiments of the present disclosure.

In a preferred embodiment, the serotype 11A saccharide conjugates of the present invention comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or about 1.0 mM glycerol per mM serotype 11A polysaccharide. In a preferred embodiment, the serotype 11A saccharide conjugates of the present invention comprise at least 0.2, 0.3 or 0.4 mM glycerol per mM serotype 11A polysaccharide. In a preferred embodiment, the serotype 11A saccharide conjugates of the present invention comprise at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or about 0.9 mM glycerol per mM serotype 11A polysaccharide and less than about 1.0 mM glycerol per mM serotype 11A polysaccharide. In a preferred embodiment, the serotype 11A saccharide conjugates of the present invention comprise at least 0.3, 0.4, 0.5, 0.6 or about 0.7 mM glycerol per mM serotype 11A polysaccharide and less than about 0.8 mM glycerol per mM serotype 11A polysaccharide. Any of the above numbers are contemplated as embodiments of the present disclosure.

Another way to characterize the glycoconjugates of the present invention is by the number of lysine residues in the carrier protein (e.g., CRM ₁₉₇ ) that are conjugated to the sugar, which can be characterized as the extent of conjugated lysine (degree of conjugation). Due to the covalent attachment to the polysaccharide, evidence of lysine modification of the carrier protein can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the present invention is 2-15, 2-13, 2-10, 2-8, 2-6, 2-5, 2-4, 3-15, 3-13, 3-10, 3-8, 3-6, 3-5, 3-4, 5-15, 5-10, 8-15, 8-12, 10-15, or 10-12. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the invention is 4 to 7. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates of the present invention can also be characterized by the ratio of saccharide to carrier protein (weight/weight). In some embodiments, the ratio of polysaccharide to carrier protein (w/w) in the glycoconjugate is 0.5-3 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the ratio of saccharide to carrier protein (w/w) is 0.5-2.0, 0.5-1.5, 0.8-1.2, 0.5-1.0, 1.0-1.5, or 1.0-2.0. In further embodiments, the ratio of saccharide to carrier protein (w/w) is 0.8-1.2. In a preferred embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is 0.9-1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates and immunogenic compositions of the present invention may contain free sugars that are not covalently conjugated to the carrier protein but are still present in the glycoconjugate composition. The free sugars may be non-covalently associated with (i.e., non-covalently bound, attached, or embedded in) the glycoconjugate.

In a preferred embodiment, the glycoconjugate comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 25% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 20% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 15% free polysaccharide compared to the total amount of polysaccharide.

The glycoconjugates can also be characterized by their molecular size distribution ( _Kd ). The relative molecular size distribution of the conjugates can be determined using size exclusion chromatography media (CL-4B). Size exclusion chromatography (SEC) is used in gravity-feed columns to characterize the molecular size distribution of the conjugates. Large molecules, which are excluded from the pores in the media, elute more rapidly than small molecules. The column eluate is collected using a fraction collector. The fractions are detected colorimetrically by saccharide assay. To determine _Kd , the column is calibrated to establish the fraction at which molecules are completely excluded ( _V0 ), ( _Kd = 0), and the fraction representing maximum retention ( _Vi ), ( _Kd = 1). The fraction that achieves a given sample property ( _Ve ) is related to _Kd by the expression _Kd = ( _Ve - _V0 )/( _Vi - _V0 ).

In a preferred embodiment, at least 30% of the glycoconjugates in the CL-4B column have a _Kd lower than or equal to 0.3. In a preferred embodiment, at least 40% of the glycoconjugates in the CL-4B column have a _Kd lower than or equal to 0.3. In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of the glycoconjugates in the CL-4B column have a _Kd lower than or equal to 0.3. In a preferred embodiment, at least 60% of the glycoconjugates in the CL-4B column have a _Kd lower than or equal to 0.3. In a preferred embodiment, 50%-80% of the glycoconjugates in the CL-4B column have a _Kd lower than or equal to 0.3. In a preferred embodiment, 65%-80% of the glycoconjugates in the CL-4B column have a _Kd lower than or equal to 0.3.

The frequency of sugar chains attached to lysine residues on the carrier protein is another parameter that characterizes the glycoconjugates of the present invention. For example, in some embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs every 4 sugar repeating units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once every 10 sugar repeating units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once every 15 sugar repeating units of the polysaccharide. In a further embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once every 25 sugar repeating units of the polysaccharide.

In conventional embodiments, the carrier protein is CRM ₁₉₇ and the covalent bond between CRM ₁₉₇ and the polysaccharide via the eTEC spacer occurs at least once per every 4, 10, 15 or 25 sugar repeating units of the polysaccharide.

In other embodiments, every 5-10 saccharide repeat units, every 2-7 saccharide repeat units, every 3-8 saccharide repeat units, every 4-9 saccharide repeat units, every 6-11 saccharide repeat units, every 7-12 saccharide repeat units, every 8-13 saccharide repeat units, every 9-14 saccharide repeat units, every 10-15 saccharide repeat units, every 2-6 saccharide repeat units, every 3-7 saccharide repeat units, every 4-8 saccharide repeat units, every 6-10 saccharide repeat units, every 7-11 saccharide repeat units, every 8-12 saccharide repeat units, every 9-13 saccharide repeat units, every 10-14 saccharide repeat units, every 10-20 saccharide repeat units, every 4-25 saccharide repeat units, or every 2-25 saccharide repeat units of the conjugate comprises at least one covalent bond between the carrier protein and the saccharide. In conventional embodiments, the carrier protein is CRM ₁₉₇ .

In another embodiment, there is at least one bond between the carrier protein and the saccharide for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 saccharide repeating units of the polysaccharide. In one embodiment, the carrier protein is CRM ₁₉₇ . Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

1.4 Combinations of the Glycoconjugates of the Present Invention

In one embodiment, the immunogenic composition of the invention comprises any of the glycoconjugates disclosed herein.

1.4.1 Combinations of Glycoconjugates

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate from S. pneumoniae serotype 9V.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate from each of the following two S. pneumoniae serotypes: 9V and 4, 9V and 6B, 9V and 14, 9V and 18C, 9V and 19F, or 9V and 23F.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate of each of the seven following S. pneumoniae serotypes: 9V, 4, 6B, 14, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate of each of the eight following S. pneumoniae serotypes: 9V, 1, 4, 6B, 14, 18C, 19F and 23F; 9V, 4, 5, 6B, 14, 18C, 19F and 23F; 9V, 4, 6B, 7F, 14, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate of each of the 10 following S. pneumoniae serotypes: 9V, 1, 5, 4, 6B, 7F, 14, 18C, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate of each of the 11 following S. pneumoniae serotypes: 9V, 1, 4, 5, 6A, 6B, 7F, 14, 18C, 19F and 23F; 9V, 1, 4, 5, 6B, 7F, 14, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate of each of the 12 following S. pneumoniae serotypes: 9V, 1, 4, 5, 6A, 6B, 7F, 14, 18C, 19A, 19F and 23F.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate of each of the 13 following S. pneumoniae serotypes: 9V, 1, 3, 4, 5, 6A, 6B, 7F, 14, 18C, 19A, 19F and 23F.

1.4.2 Additional glycoconjugate combinations

In one embodiment, any of the immunogenic compositions defined above in 1.4.1 further comprises at least one saccharide conjugate of S. pneumoniae serotype 15B.

In one embodiment, any of the immunogenic compositions defined above in 1.4.1 further comprises at least one saccharide conjugate of S. pneumoniae serotype 22F.

In one embodiment, any of the immunogenic compositions defined above in 1.4.1 further comprises at least one saccharide conjugate of S. pneumoniae serotype 33F.

In one embodiment, any of the immunogenic compositions defined above in 1.4.1 further comprises at least one saccharide conjugate of S. pneumoniae serotype 8.

In one embodiment, any immunogenic composition defined above in 1.4.1 further comprises at least one saccharide conjugate of S. pneumoniae serotype 10A.

In one embodiment, any immunogenic composition defined above in 1.4.1 further comprises at least one saccharide conjugate of S. pneumoniae serotype 11A.

In one embodiment, any of the immunogenic compositions defined above in 1.4.1 further comprises at least one saccharide conjugate of S. pneumoniae serotype 12F.

In one embodiment, any immunogenic composition defined in 1.4.1 above further comprises at least one saccharide conjugate of each of the two following S. pneumoniae serotypes:

15B and 22F,

15B and 33F,

15B and 12F,

15B and 10A,

15B and 11A,

15B and 8,

22F and 33F,

22F and 12F,

22F and 10A,

22F and 11A,

22F and 8,

33F and 12F,

33F and 10A,

33F and 11A,

33F and 8,

12F and 10A,

12F and 11A,

12F and 8,

10A and 11A,

10A and 8, or

11A and 8.

In one embodiment, any immunogenic composition defined in 1.4.1 above further comprises at least one saccharide conjugate of each of the three following S. pneumoniae serotypes:

15B and 22F and 33F,

15B and 22F and 12F,

15B and 22F and 10A,

15B and 22F and 11A,

15B and 22F and 8,

15B and 33F and 12F,

15B and 33F and 10A,

15B and 33F and 11A,

15B and 33F and 8,

15B and 12F and 10A,

15B and 12F and 11A,

15B and 12F and 8,

15B and 10A and 11A,

15B and 10A and 8,

15B and 11A and 8,

22F and 33F and 12F,

22F and 33F and 10A,

22F and 33F and 11A,

22F and 33F and 8,

22F and 12F and 10A,

22F, 12F, and 11A,

22F and 12F and 8,

22F and 10A and 11A,

22F and 10A and 8,

22F and 11A and 8,

33F and 12F and 10A,

33F, 12F, and 11A,

33F and 12F and 8,

33F and 10A and 11A,

33F and 10A and 8,

33F and 11A and 8,

12F, 10A, and 11A,

12F and 10A and 8,

12F and 11A and 8, or

10A and 11A and 8.

In one embodiment, any immunogenic composition defined in 1.4.1 above further comprises at least one saccharide conjugate of each of the four following S. pneumoniae serotypes:

15B and 22F and 33F and 12F,

15B and 22F and 33F and 10A,

15B and 22F and 33F and 11A,

15B and 22F and 33F and 8,

15B and 22F and 12F and 10A,

15B and 22F and 12F and 11A,

15B and 22F and 12F and 8,

15B and 22F and 10A and 11A,

15B and 22F and 10A and 8,

15B and 22F and 11A and 8,

15B and 33F and 12F and 10A,

15B and 33F and 12F and 11A,

15B and 33F and 12F and 8,

15B and 33F and 10A and 11A,

15B and 33F and 10A and 8,

15B and 33F and 11A and 8,

15B and 12F and 10A and 11A,

15B and 12F and 10A and 8,

15B and 12F and 11A and 8,

15B and 10A and 11A and 8,

22F and 33F and 12F and 10A,

22F and 33F and 12F and 11A,

22F and 33F and 12F and 8,

22F and 33F and 10A and 11A,

22F and 33F and 10A and 8,

22F and 33F and 11A and 8,

22F and 12F and 10A and 11A,

22F and 12F and 10A and 8,

22F and 12F and 11A and 8,

22F and 10A and 11A and 8,

33F and 12F and 10A and 11A,

33F and 12F and 10A and 8,

33F and 12F and 11A and 8,

33F and 10A and 11A and 8, or

12F and 10A and 11A and 8.

In one embodiment, any of the immunogenic compositions defined above in 1.4.1 further comprises at least one saccharide conjugate of each of the five following S. pneumoniae serotypes:

15B and 22F and 33F and 12F and 10A,

15B and 22F and 33F and 12F and 11A,

15B and 22F and 33F and 12F and 8,

15B and 22F and 33F and 10A and 11A,

15B and 22F and 33F and 10A and 8,

15B and 22F and 33F and 11A and 8,

15B and 22F and 12F and 10A and 11A,

15B and 22F and 12F and 10A and 8,

15B and 22F and 12F and 11A and 8,

15B and 22F and 10A and 11A and 8,

15B and 33F and 12F and 10A and 11A,

15B and 33F and 12F and 10A and 8,

15B and 33F and 12F and 11A and 8,

15B and 33F and 10A and 11A and 8,

15B and 12F and 10A and 11A and 8,

22F and 33F and 12F and 10A and 11A,

22F and 33F and 12F and 10A and 8,

22F and 33F and 12F and 11A and 8,

22F and 33F and 10A and 11A and 8,

22F and 12F and 10A and 11A and 8, or

33F and 12F and 10A and 11A and 8.

In one embodiment, any immunogenic composition defined in 1.4.1 above further comprises at least one saccharide conjugate of each of the six following S. pneumoniae serotypes:

15B and 22F and 33F and 12F and 10A and 11A,

15B and 22F and 33F and 12F and 10A and 8,

15B and 22F and 33F and 12F and 11A and 8,

15B and 22F and 33F and 10A and 11A and 8,

15B and 22F and 12F and 10A and 11A and 8,

15B and 33F and 12F and 10A and 11A and 8, or

22F and 33F and 12F and 10A and 11A and 8.

In one embodiment, any immunogenic composition defined in 1.4.1 above additionally comprises at least one saccharide conjugate of each of the seven following S. pneumoniae serotypes: 15B and 22F and 33F and 12F and 10A and 11A and 8.

In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate of S. pneumoniae serotype 2.

In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate of S. pneumoniae serotype 17F.

In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate of S. pneumoniae serotype 20.

In one embodiment, any of the above immunogenic compositions further comprises a saccharide conjugate of S. pneumoniae serotype 15C.

Preferably, all glycoconjugates of the above immunogenic compositions are individually conjugated to the carrier protein.

In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 9V is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 22F is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 33F is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 15B is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 12F is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 10A is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 11A is conjugated to CRM ₁₉₇ . In one embodiment of the above immunogenic composition, a saccharide conjugate from S. pneumoniae serotype 8 is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from S. pneumoniae serotypes 4, 6B, 14, 18C, 19F and 23F are conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from S. pneumoniae serotypes 1, 5 and 7F are conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from S. pneumoniae serotypes 6A and 19A are conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from S. pneumoniae serotype 3 is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from S. pneumoniae serotype 2 is conjugated to CRM ₁₉₇ . In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 17F is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 20 is conjugated to CRM _197. In one embodiment of the above immunogenic composition, a saccharide conjugate from Streptococcus pneumoniae serotype 15C is conjugated to CRM ₁₉₇ .

In one embodiment, all of the glycoconjugates of the immunogenic compositions described above are individually conjugated to CRM ₁₉₇ .

In one embodiment, the saccharide conjugate from S. pneumoniae serotype 9V of any of the above immunogenic compositions is conjugated solely to PD.

In one embodiment, the saccharide conjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogenic compositions are individually conjugated to PD.

In one embodiment, the saccharide conjugate from S. pneumoniae serotype 18C of any of the above immunogenic compositions is solely conjugated to TT.

In one embodiment, the saccharide conjugate from S. pneumoniae serotype 19F of any of the above immunogenic compositions is conjugated to DT.

In one embodiment, the saccharide conjugates from S. pneumoniae serotype 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F of any of the above immunogenic compositions are individually conjugated to PD, the saccharide conjugate from S. pneumoniae serotype 18C is conjugated to TT, and the saccharide conjugate from S. pneumoniae serotype 19F is conjugated to DT.

In one embodiment, the immunogenic composition comprises 7-24 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition comprises saccharide conjugates of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 different serotypes.

In one embodiment, the immunogenic composition comprises 7-20 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition comprises saccharide conjugates of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition comprises saccharide conjugates of 16 or 20 different serotypes.

In one embodiment, the immunogenic composition is an 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 14-, 15-, 16-, 17-, 18-, or 19-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

In one embodiment, the immunogenic composition of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises saccharide conjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises conjugated saccharides of S. pneumoniae from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the immunogenic composition of the invention comprises conjugated saccharides of S. pneumoniae from serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

In one embodiment, the glycoconjugates of the immunogenic composition of the invention consist of glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In one embodiment, the glycoconjugates of the immunogenic composition of the invention consist of glycoconjugates from serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In one embodiment, the glycoconjugates of the immunogenic composition of the invention consist of glycoconjugates from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In one embodiment, the glycoconjugates of the immunogenic composition of the invention consist of glycoconjugates from 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.

Preferably, all glycoconjugates of the immunogenic composition of the present invention are individually conjugated to a carrier protein. In one embodiment, the glycoconjugates of the above immunogenic composition are individually conjugated to CRM ₁₉₇ .

1.4.3 Further glycoconjugate combinations

In one embodiment, any immunogenic composition defined above in 1.4.1 or 1.4.2 does not comprise capsular saccharides of S. pneumoniae serotype 9N.

In one embodiment, any immunogenic composition defined in 1.4.1 or 1.4.2 above does not comprise capsular saccharides of S. pneumoniae serotype 9A.

In one embodiment, any immunogenic composition defined in 1.4.1 or 1.4.2 above does not comprise capsular saccharides of S. pneumoniae serotype 9L.

In one embodiment, any immunogenic composition defined in 1.4.1 or 1.4.2 above does not comprise capsular saccharides of S. pneumoniae serotypes 9N and 9A.

In one embodiment, any immunogenic composition defined in 1.4.1 or 1.4.2 above does not comprise capsular saccharides of S. pneumoniae serotypes 9N and 9L.

In one embodiment, any immunogenic composition defined in 1.4.1 or 1.4.2 above does not comprise capsular saccharides of S. pneumoniae serotypes 9A and 9L.

In one embodiment, any immunogenic composition defined in 1.4.1 or 1.4.2 above does not comprise capsular saccharides of S. pneumoniae serotypes 9N, 9A and 9L.

After the capsular polysaccharide is conjugated to the carrier protein, the glycoconjugate is purified by various techniques (to enrich the amount of polysaccharide-carrier protein conjugate). These techniques include concentration/diafiltration, precipitation/elution, column chromatography, and depth filtration. See, for example, U.S. Application Publication Nos. 2007/0184072 and WO 2008/079653. After the individual glycoconjugates are purified, they are mixed to formulate the immunogenic composition of the present invention.

2. Dosage of the immunogenic composition

2.1 Amount of polysaccharides

The amount of glycoconjugate selected in each dose is that which induces an immunoprotective response in a typical vaccinee without significant adverse side effects. This amount will vary depending on the specific immunogen used and how it is presented.

The amount of a particular saccharide conjugate in an immunogenic composition can be calculated based on the total polysaccharide amount of the conjugate (conjugated and unconjugated). For example, in a 100 μg polysaccharide dose, a saccharide conjugate with 20% free polysaccharide has approximately 80 μg conjugated polysaccharide and approximately 20 μg unconjugated polysaccharide. The amount of saccharide conjugate can vary depending on the Streptococcus serotype. The sugar concentration can be determined by uronic acid assay.

The "immunogenic amount" of the different polysaccharide components in the immunogenic composition can vary, and each can comprise about 1.0 μg, about 2.0 μg, about 3.0 μg, about 4.0 μg, about 5.0 μg, about 6.0 μg, about 7.0 μg, about 8.0 μg, about 9.0 μg, about 10.0 μg, about 15.0 μg, about 20.0 μg, about 30.0 μg, about 40.0 μg, about 50.0 μg, about 60.0 μg, about 70.0 μg, about 80.0 μg, about 90.0 μg, or about 100.0 μg of any particular polysaccharide antigen.

Typically, for a given serotype, each dose comprises 0.1 μg-100 μg polysaccharide, particularly 0.5 μg-20 μg, more particularly 1 μg-10 μg, and even more particularly 2 μg-5 μg.Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

In one embodiment, for a given serotype, each dose comprises 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg or 20 μg of polysaccharide.

2.2 Amount of carrier

Typically, each dose contains 5 μg-150 μg of carrier protein, particularly 10 μg-100 μg of carrier protein, more particularly 15 μg-100 μg of carrier protein, more particularly 25-75 μg of carrier protein, more particularly 30 μg-70 μg of carrier protein, more particularly 30-60 μg of carrier protein, more particularly 30 μg-50 μg of carrier protein and more particularly 40-60 μg of carrier protein. In one embodiment, the carrier protein is CRM ₁₉₇ .

In one embodiment, each dose comprises about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg. In one embodiment, the carrier protein is CRM ₁₉₇ .

3 Further antigens

The immunogenic composition of the present invention comprises a conjugated Streptococcus pneumoniae saccharide antigen (glycoconjugate). It may further comprise antigens from other pathogens (particularly from bacteria and/or viruses). Preferred further antigens are selected from the group consisting of: diphtheria toxoid (D), tetanus toxoid (T), pertussis antigen (P) (typically acellular (Pa)), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis A virus (HAV) antigen, conjugated Haemophilus influenzae type b capsular saccharide (Hib), and inactivated poliovirus vaccine (IPV).

In one embodiment, the immunogenic composition of the present invention comprises D-T-Pa. In one embodiment, the immunogenic composition of the present invention comprises D-T-Pa-Hib, D-T-Pa-IPV, or D-T-Pa-HBsAg. In one embodiment, the immunogenic composition of the present invention comprises D-T-Pa-HBsAg-IPV or D-T-Pa-HBsAg-Hib. In one embodiment, the immunogenic composition of the present invention comprises D-T-Pa-HBsAg-IPV-Hib.

Pertussis antigens: Bordetella pertussis causes pertussis. The pertussis antigens in the vaccine are either cellular (whole cells, in the form of inactivated B. pertussis cells) or acellular. The preparation of cellular pertussis antigens is well documented (for example, they can be obtained by heat-inactivating a Phase I culture of B. pertussis). However, the present invention preferably uses acellular antigens. When acellular antigens are used, preferably one, two, or (preferably) three of the following antigens are used: (1) detoxified pertussis toxin (pertussis toxoid, or PT); (2) filamentous hemagglutinin (FHA); and (3) pertactin (also known as the 69 kilodalton outer membrane protein). FHA and pertactin may be treated with formaldehyde prior to use in accordance with the present invention. PT is preferably detoxified by treatment with formaldehyde and/or glutaraldehyde. The acellular pertussis antigens are preferably adsorbed onto one or more aluminum salt adjuvants. Alternatively, they may be added in a non-adsorbed state. When pertactin is added, it is preferably already adsorbed on aluminum hydroxide adjuvant. PT and FHA may be adsorbed on aluminum hydroxide adjuvant or aluminum phosphate. Most preferably, all of PT, FHA, and pertactin are adsorbed on aluminum hydroxide.

Inactivated poliovirus vaccine: Poliovirus causes polio. Instead of using an oral poliovirus vaccine, a preferred embodiment of the present invention uses IPV. Before administration to a patient, the poliovirus must be inactivated, and this can be achieved by treatment with formaldehyde. Poliomyelitis can be caused by one of three types of poliovirus. These three types are similar and cause the same symptoms, but their antigenicity is different, and infection with one type does not protect against infection with the other types. Therefore, it is preferred to use three poliovirus antigens in the present invention: poliovirus type 1 (e.g., Mahoney strain), poliovirus type 2 (e.g., MEF-1 strain), and poliovirus type 3 (e.g., Saukett strain). The viruses are preferably grown, purified, and inactivated individually, and then combined to provide a large trivalent mixture for use in the present invention.

Diphtheria toxoid: Corynebacterium diphtheriae causes diphtheria. The diphtheria toxin can be treated (e.g., using formalin or formaldehyde) to remove toxicity while retaining the ability to induce specific anti-toxin antibodies after injection. These diphtheria toxoids are used in diphtheria vaccines. Preferred diphtheria toxoids are those prepared by formaldehyde treatment. The diphtheria toxoid can be obtained by growing Corynebacterium diphtheriae in a growth medium, followed by treatment with formaldehyde, ultrafiltration, and precipitation. The toxoid material can then be processed through a procedure including sterile filtration and/or dialysis. The diphtheria toxoid is preferably adsorbed onto an aluminum hydroxide adjuvant.

Tetanus Toxoid: Clostridium tetani causes tetanus. Tetanus toxin can be processed to provide a protective toxoid. Such toxoids are used in tetanus vaccines. Preferred tetanus toxoids are those prepared by formaldehyde treatment. The tetanus toxoid can be obtained by growing the bacteria in a growth medium, followed by formaldehyde treatment, ultrafiltration, and precipitation. The material is then processed through procedures including sterile filtration and/or dialysis.

Hepatitis A virus antigen: Hepatitis A virus (HAV) is one of the known agents that cause viral hepatitis. Preferred HAV components are based on inactivated virus, and inactivation can be achieved by formalin treatment.

Hepatitis B virus (HBV) is one of the known agents of viral hepatitis. The main component of the capsid is a protein called HBV surface antigen, or more commonly HBsAg, which is typically a 226-amino acid polypeptide with a molecular weight of ~24 kDa. All existing hepatitis B virus vaccines contain HBsAg, and when this antigen is administered to normal vaccinees, it stimulates the production of anti-HBsAg antibodies, which protect against HBV infection.

For vaccine production, HBsAg is prepared in two ways: by purifying the antigen in the form of particles from the plasma of chronic hepatitis B virus carriers, or by expressing the protein via recombinant DNA methods (e.g., recombinant expression in yeast cells). Unlike native HBsAg (i.e., in plasma-purified products), yeast-expressed HBsAg is typically non-glycosylated and is the most preferred form of HBsAg for use in the present invention.

Conjugated Haemophilus influenzae type b antigens: Haemophilus influenzae type b (Hib) causes bacterial meningitis. Hib vaccines are typically based on capsular saccharide antigens, the preparation of which is well documented. Hib saccharides can be conjugated to carrier proteins to enhance their immunogenicity, particularly in children. Typical carrier proteins are tetanus toxoid, diphtheria toxoid, CRM197, Haemophilus influenzae protein D and the outer membrane protein complex from meningococcal serotype B. The sugar portion of the conjugate may comprise fragments of full-length polyribosylribitol phosphate and/or full-length PRP prepared from the Hib bacterium. The Hib conjugate may or may not be adsorbed to an aluminum salt adjuvant.

In one embodiment, the immunogenic composition of the invention further comprises a conjugated Neisseria meningitidis serotype Y capsular saccharide (MenY) and/or a conjugated Neisseria meningitidis serotype C capsular saccharide (MenC).

In one embodiment, the immunogenic composition of the invention further comprises a conjugated Neisseria meningitidis serotype A capsular saccharide (MenA), a conjugated Neisseria meningitidis serotype W135 capsular saccharide (MenW135), a conjugated Neisseria meningitidis serotype Y capsular saccharide (MenY) and/or a conjugated Neisseria meningitidis serotype C capsular saccharide (MenC).

In one embodiment, the immunogenic composition of the invention further comprises conjugated Neisseria meningitidis serotype W135 capsular saccharide (MenW135), conjugated Neisseria meningitidis serotype Y capsular saccharide (MenY) and/or conjugated Neisseria meningitidis serotype C capsular saccharide (MenC).

4 Adjuvants

In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one adjuvant (e.g., 1, 2, or 3 adjuvants). The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An antigen may function primarily as a delivery system, primarily as an immunomodulator, or possess both of these strong characteristics. Suitable adjuvants include those suitable for use in mammals, including humans.

Examples of known suitable delivery system-type adjuvants that can be used in humans include, but are not limited to, aluminum (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

In one embodiment, the immunogenic composition disclosed herein comprises an aluminum salt (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide). In a preferred embodiment, the immunogenic composition disclosed herein comprises aluminum phosphate or aluminum hydroxide as an adjuvant. In one embodiment, the immunogenic composition disclosed herein comprises 0.1 mg/mL to 1 mg/mL or 0.2 mg/mL to 0.3 mg/mL of aluminum in the form of aluminum phosphate. In one embodiment, the immunogenic composition disclosed herein comprises approximately 0.25 mg/mL of aluminum in the form of aluminum phosphate.

Examples of suitable immunomodulatory adjuvants known to be useful in humans include, but are not limited to, Aquilla tree (QS21, Quil A), TLR4 agonists such as MPL (monophosphoryl lipid A), 3DMPL (3-O-deacetylated MPL) or GLA-AQ, LT/CT mutants, cytokines such as various interleukins (e.g., IL-2, IL-12) or GM-CSF, and the like.

Examples of suitable immunomodulatory adjuvants known to be useful in humans that have both delivery and immunomodulatory characteristics include, but are not limited to, ISCOMS (see, e.g., et al. (1998) J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630, WO 98/36772, WO 00/41720, WO 2006/134423 and WO 2007/026190) or GLA-EM, which is a combination of a TLR4 agonist and an oil-in-water emulsion.

For veterinary applications (including but not limited to animal experiments), Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), Emulsigen, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-demuramoyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamido-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphooxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeletal protein (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion, can be used.

Further examples of adjuvants for enhancing the efficacy of pneumococcal vaccines as disclosed herein include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (see below) or bacterial cell wall components), such as (a) SAF, containing 10% squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, microfluidized into a submicron emulsion or vortexed to produce an emulsion with larger particle size, and (b) RIBI ^™ Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT), containing 2% squalane, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall scaffold protein (CWS), preferably MPL + CWS (DETOX ^™ ); (2) saponin adjuvants, such as QS21, STIMULON ^™ (Cambridge Bioscience, Worcester, MA), (Isconova, Sweden) or (Commonwealth Serum Laboratories, Australia), or particles derived therefrom such as ISCOMs (immunostimulatory complexes), which may be free of additional detergents (e.g., WO 00/07621); (3) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO 99/44636)), interferons (e.g., interferon-γ), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacetylated MPL (3dMPL) (see, e.g., GB-2220221, EP0689454), optionally substantially free of aluminum when used with Streptococcus pneumoniae saccharides (see, e.g., WO 00/56358); (6) combinations of 3dMPL with, e.g., QS21 and/or oil-in-water emulsions (see, e.g., EP0835318, EP0735898, EP0761231); (7) polyoxyethylene ethers or polyoxyethylene esters (see, e.g., WO 99/52549); (8) combinations of polyoxyethylene sorbitan ester surfactants with octoxynol (WO 01/21207), or a combination of a polyoxyethylene alkyl ether or ester surfactant with at least one additional nonionic surfactant such as octylphenol polyether (WO 01/21152); (9) saponin and immunostimulatory oligonucleotides (e.g. CpG oligonucleotides) (WO 00/62800); (10) immunostimulatory agents and metal salt particles (see, for example, WO 00/23105); (11) saponin and oil-in-water emulsions, for example, WO 99/11241; (12) saponin (e.g. QS21) + 3dMPL + IM2 (optionally + sterol), for example, WO 98/57659; (13) other substances that act as immunostimulatory agents to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphate)-ethylamine MTP-PE), etc.

In one embodiment of the present invention, the immunogenic compositions disclosed herein comprise CpG oligonucleotides as adjuvants. The CpG oligonucleotides used in the present invention are referred to as immunostimulatory CpG oligodeoxynucleotides (CpG ODNs), and therefore these terms can be used interchangeably unless otherwise specified. Immunostimulatory CpG oligodeoxynucleotides contain one or more immunostimulatory CpG motifs, which are unmethylated cytosine-guanine dinucleotides, optionally within certain preferred base environments. The methylation state of a CpG immunostimulatory motif generally refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide containing a 5' unmethylated cytosine linked to a 3' guanine by a phosphate bond, and which activates the immune system by binding to Toll-like receptor 9 (TLR-9). In another embodiment, the immunostimulatory oligonucleotide may contain one or more methylated CpG dinucleotides, which activate the immune system through TLR9, but not as strongly as if the CpG motif were unmethylated. CpG immunostimulatory oligonucleotides may comprise one or more palindromic sequences, which in turn may surround CpG dinucleotides. CpG oligonucleotides have been described in numerous issued patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, and 6,339,068.

In one embodiment of the present invention, the immunogenic composition disclosed herein comprises any CpG oligonucleotide described on page 3, line 22 to page 12, line 36 of WO 2010/125480.

Different classes of CpG immunostimulatory oligonucleotides have been identified. These are referred to as classes A, B, C, and P and are described in detail in WO 2010/125480, page 3, line 22 to page 12, line 36. The methods of the present invention include the use of these different classes of CpG immunostimulatory oligonucleotides.

In one embodiment of the present invention, the immunogenic composition disclosed herein comprises a CpG oligonucleotide of class A. In one embodiment of the present invention, the immunogenic composition disclosed herein comprises a CpG oligonucleotide of class B.

The B-class oligonucleotide sequences of the present invention are those described in the above extensive discussion and in published WO 96/02555, WO 98/18810 and in U.S. Patent Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116 and 6,339,068. Exemplary sequences include, but are not limited to, those disclosed in these latter patent applications and patents.

In one embodiment, a "class B" CpG oligonucleotide of the invention has the following nucleic acid sequence:

5'TCGTCGTTTTTCGGTGCTTTT 3' (SEQ ID NO: 1), or

5'TCGTCGTTTTTCGGTCGTTTT 3' (SEQ ID NO: 2), or

5'TCGTCGTTTTGTCGTTTTGTCGTT 3' (SEQ ID NO: 3), or

5’TCGTCGTTTCGTCGTTTTGTCGTT 3’(SEQ ID NO:4), or

5'TCGTCGTTTTGTCGTTTTTTTCGA 3' (SEQ ID NO: 5).

In any of these sequences, all linkages may be phosphorothioate bonds. In another embodiment, in any of these sequences, one or more linkages may be phosphodiester, preferably between the "C" and "G" of the CpG motif to create a semi-soft CpG oligonucleotide. In any of these sequences, ethyl-uridine or a halogen may replace the 5'T; examples of halogen substitutions include, but are not limited to, bromo-uridine or iodo-uridine substitutions.

Some non-limiting examples of Class B oligonucleotides include:

5’T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3’(SEQ ID NO:6), or

5’T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3’(SEQ ID NO:7), or

5’T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3’(SEQ ID NO:8), or

5’T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3’(SEQ ID NO:9), or

5’T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A 3’ (SEQ ID NO: 10).

Wherein “*” refers to a phosphorothioate bond.

In one embodiment of the present invention, the immunogenic composition disclosed herein comprises a C-class CpG oligonucleotide.

In one embodiment of the present invention, the immunogenic composition disclosed herein comprises a P-type CpG oligonucleotide.

In one embodiment, the oligonucleotide comprises at least one phosphorothioate bond. In another embodiment, the internucleotide connections of all oligonucleotides are phosphorothioate bonds. In another embodiment, the oligonucleotide comprises at least one phosphodiester-like connection. In another embodiment, the phosphodiester-like connection is a phosphodiester bond. In another embodiment, a lipophilic group is conjugated to the oligonucleotide. In one embodiment, the lipophilic group is cholesterol.

In one embodiment, all internucleotide connections of the CpG oligonucleotides disclosed herein are phosphodiester bonds ("soft" oligonucleotides, as described in WO 2007/026190). In another embodiment, the CpG oligonucleotides of the present invention are made resistant to degradation (e.g., stabilized). "Stabilized oligonucleotides" refer to oligonucleotides that are relatively resistant to degradation in vivo (e.g., by exo- or endo-nucleases). Nucleic acid stabilization can be accomplished by backbone modification. Oligonucleotides with phosphorothioate connections provide maximum activity and protect the oligonucleotide from degradation by intracellular endo- and exo-nucleases.

Immunostimulatory oligonucleotide can have chimeric main chain, and it has the combination that phosphodiester is connected with phosphorothioate.For purpose of the present invention, chimeric main chain refers to the main chain of part stabilization, wherein at least one internucleotide connection is phosphodiester or phosphodiester sample, and wherein at least one other internucleotide connection is stabilization internucleotide connection, and the connection of wherein said at least one phosphodiester or phosphodiester sample is different from described at least one stabilization connection.When phosphodiester connection is preferably positioned in the CpG motif, this molecule is referred to as " semi-soft ", as described in WO 2007/026190.

Other modified oligonucleotides include phosphodiester, phosphorothioate, methylphosphonate, methylphosphorothioate, phosphorodithioate and/or p-ethoxy linkages.

Mixed backbone-modified ODNs can be synthesized as described in WO 2007/026190.

In some embodiments, the CpG oligonucleotide has a length of 6 to 100 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 30 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 100 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 30 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 100 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 30 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 100 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 30 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 6 to 100 nucleotides. In some embodiments, the CpG oligonucleotide has a length of 8 ...

In one embodiment, the CpG oligonucleotide disclosed herein comprises substitutions or modifications, such as those described on pages 134-137 of WO 2007/026190, in bases and/or sugars.

In one embodiment, the CpG oligonucleotides of the invention are chemically modified. Examples of chemical modifications are known to those skilled in the art, such as those described in Uhlmann et al. (1990) Chem. Rev. 90: 543; S. Agrawal, Ed., Humana Press, Totowa, USA 1993; Crooke et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36: 107-129 and Hunziker et al. (1995) Mod. Synth. Methods 7: 331-417. The oligonucleotides of the invention may have one or more modifications, wherein each modification is located at a specific phosphodiester internucleotide linkage and/or at a specific β-D-ribose unit and/or at a specific natural nucleobase position compared to an oligonucleotide of the same sequence consisting of natural DNA or RNA.

In some embodiments of the invention, the CpG-containing nucleic acid may be simply mixed with an immunogenic carrier according to methods known to those skilled in the art (see, eg, WO 03/024480).

In a specific embodiment of the present invention, any immunogenic composition disclosed herein comprises 2 μg-100 mg of CpG oligonucleotide, preferably 0.1 mg-50 mg of CpG oligonucleotide, preferably 0.2 mg-10 mg of CpG oligonucleotide, preferably 0.3 mg-5 mg of CpG oligonucleotide, preferably 0.3 mg-5 mg of CpG oligonucleotide, more preferably 0.5 mg-2 mg of CpG oligonucleotide, more preferably 0.75 mg-1.5 mg of CpG oligonucleotide. In a preferred embodiment, any immunogenic composition disclosed herein comprises approximately 1 mg of CpG oligonucleotide.

5 Preparation

The immunogenic compositions of the present invention can be formulated in liquid form (i.e., solution or suspension) or in lyophilized form. Liquid formulations can advantageously be administered directly from their packaging and are therefore ideal for injection, without the need for reconstitution in an aqueous medium, whereas the lyophilized compositions of the present invention require reconstitution in a liquid medium.

The immunogenic compositions of the present invention can be formulated using methods recognized in the art. For example, each pneumococcal conjugate can be formulated with a physiologically acceptable carrier to prepare the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

The present invention provides immunogenic compositions comprising a combination of any of the glycoconjugates disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

In one embodiment, the immunogenic composition of the invention is in liquid form, preferably in aqueous liquid form.

The immunogenic compositions of the present invention may comprise one or more buffers, salts, divalent cations, nonionic detergents, cryoprotectants such as sugars, and antioxidants such as free radical scavengers or chelating agents, or any combination thereof.

In one embodiment, the immunogenic composition of the present invention comprises a buffer. In one embodiment, the pKa of the buffer is about 3.5 to about 7.5. In some embodiments, the buffer is phosphate, succinate, histidine, or citrate. In certain embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM. In a specific embodiment, the final concentration of the succinate buffer is about 5 mM.

In one embodiment, the immunogenic composition of the present invention comprises a salt. In some embodiments, the salt is selected from magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a specific embodiment, the salt is sodium chloride. In a specific embodiment, the immunogenic composition of the present invention comprises 150 mM sodium chloride.

In one embodiment, the immunogenic composition of the present invention comprises a surfactant. In one embodiment, the surfactant is selected from the group consisting of polysorbate 20 (TWEEN ^™ 20), polysorbate 40 (TWEEN ^™ 40), polysorbate 60 (TWEEN ^™ 60), polysorbate 65 (TWEEN ^™ 65), polysorbate 80 (TWEEN ^™ 80), polysorbate 85 (TWEEN ^™ 85), TRITON ^™ N-101, TRITON ^™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyethylene glycol-660 hydroxystearate (PEG-15, Solutol H 15), polyethylene glycol-35-ricinoleate (EL), soy lecithin, and poloxamer. In a specific embodiment, the surfactant is polysorbate 80. In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.0001% to 10% polysorbate 80 by weight (w/w). In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001% to 1% polysorbate 80 by weight (w/w). In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01% to 1% polysorbate 80 by weight (w/w). In other embodiments, the final concentration of polysorbate 80 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 80 (w/w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 1% polysorbate 80 (w/w).

In certain embodiments, the pH of the immunogenic composition of the invention is 5.5-7.5, more preferably pH 5.6-7.0, even more preferably pH 5.8-6.0.

In one embodiment, the present invention provides a container filled with any of the immunogenic compositions disclosed herein. In one embodiment, the container is selected from the group consisting of a vial, a syringe, a flask, a fermentation bottle, a bioreactor, a pharmaceutical bag, a pharmaceutical canister, an ampoule, a cartridge, and a disposable pen. In certain embodiments, the container is siliconized.

In one embodiment, the container of the present invention is made of glass, metal (such as steel, stainless steel, aluminum, etc.) and/or polymer (such as thermoplastic, elastomer, thermoplastic elastomer).In one embodiment, the container of the present invention is made of glass.

In one embodiment, the present invention provides a syringe filled with any of the immunogenic compositions disclosed herein. In certain embodiments, the syringe is siliconized and/or made of glass.

Typical dosages of the immunogenic compositions of the invention are in volumes of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, and even more preferably in volumes of about 0.5 mL.

Thus, the container or syringe as defined above is filled with a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, even more preferably about 0.5 mL of any immunogenic composition defined in the present invention.

6. Ability of the immunogenic composition of the present invention to stimulate cross-reactive antibodies

In one embodiment, the immunogenic composition of the invention is capable of eliciting IgG antibodies in humans that bind to S. pneumoniae serotype 9N, 9A and/or 9L polysaccharides as determined by ELISA.

In the ELISA (enzyme-linked immunosorbent assay) method, antibodies from the serum of vaccinated subjects are incubated with a polysaccharide that has been adsorbed to a solid support. Bound antibodies are detected using an enzyme-conjugated secondary detection antibody.

In one embodiment, the ELISA assay is a standardized ELISA assay specified by WHO in “Training Manual For Enzyme Linked Immunosorbent Assay For The Quantitation Of Streptococcus Pneumoniae Serotype Specific IgG (Pn PS ELISA)” (see http://www.vaccine.uab.edu/ELISA%20protocol.pdf, accessed March 31, 2014).

This ELISA measures the presence of type-specific IgG anti-S. pneumoniae capsular polysaccharide (PS) antibodies in human serum. When diluted human serum is added to microtiter plates coated with type-specific capsular PS, antibodies specific for capsular PS bind to the plates. Plate-bound antibodies are detected using a goat anti-human IgG alkaline phosphatase-labeled antibody followed by p-nitrophenyl phosphate substrate. The optical density of the resulting colored product is proportional to the amount of anti-capsular PS antibodies present in the serum.

In one embodiment, the immunogenic composition of the invention can elicit IgG antibodies in humans at a concentration of at least 0.05 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml or 0.5 μg/ml as determined by ELISA, wherein the IgG antibodies can bind to S. pneumoniae serotype 9N polysaccharide.

In one embodiment, the immunogenic composition of the invention can elicit IgG antibodies in humans at a concentration of at least 0.05 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml or 0.5 μg/ml as determined by ELISA, wherein the IgG antibodies can bind to S. pneumoniae serotype 9A polysaccharide.

In one embodiment, the immunogenic composition of the invention can elicit IgG antibodies in humans at a concentration of at least 0.05 μg/ml, 0.1 μg/ml, 0.2 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml or 0.5 μg/ml as determined by ELISA, wherein the IgG antibodies can bind to S. pneumoniae serotype 9L polysaccharide.

In one embodiment, the immunogenic compositions of the invention are capable of eliciting functional antibodies in humans, such as antibodies capable of killing S. pneumoniae serotypes 9N, 9A, and/or 9L, as determined by an in vitro opsonophagocytosis assay (OPA) (see Example 1). In one embodiment, the immunogenic compositions of the invention are capable of eliciting functional antibodies in humans, such as antibodies capable of killing S. pneumoniae serotype 9N, as determined by an in vitro opsonophagocytosis assay (OPA). In one embodiment, the immunogenic compositions of the invention are capable of eliciting functional antibodies in humans, such as antibodies capable of killing S. pneumoniae serotype 9A, as determined by an in vitro opsonophagocytosis assay (OPA). In one embodiment, the immunogenic compositions of the invention are capable of eliciting functional antibodies in humans, such as antibodies capable of killing S. pneumoniae serotype 9L, as determined by an in vitro opsonophagocytosis assay (OPA). In one embodiment, the immunogenic compositions of the invention are capable of eliciting functional antibodies in humans, such as antibodies capable of killing S. pneumoniae serotypes 9N and 9A, as determined by an in vitro opsonophagocytosis assay (OPA). In one embodiment, the immunogenic compositions of the invention are capable of eliciting functional antibodies in humans that are capable of killing S. pneumoniae serotypes 9N and 9L as determined by an in vitro opsonophagocytosis assay (OPA). In one embodiment, the immunogenic compositions of the invention are capable of eliciting functional antibodies in humans that are capable of killing S. pneumoniae serotypes 9A and 9L as determined by an in vitro opsonophagocytosis assay (OPA).

The pneumococcal opsonophagocytosis assay (OPA), which measures the killing of S. pneumoniae cells by phagocytic effector cells in the presence of functional antibodies and complement, is considered an important surrogate for assessing the efficacy of pneumococcal vaccines.

The in vitro opsonophagocytosis assay (OPA) can be performed by incubating a mixture of S. pneumoniae cells, heat-inactivated human serum to be tested, differentiated HL-60 cells (phagocytes), and an external complement source (e.g., baby rabbit complement). During the incubation period, opsonophagocytosis occurs, and the bacterial cells coated with antibodies and complement are killed on the basis of opsonophagocytosis. The colony forming units (cfu) of surviving bacteria that escape from opsonophagocytosis are determined by plating the assay mixture. The OPA titer is defined as the reciprocal dilution that results in a 50% reduction in bacterial counts compared to a control without test serum. The OPA titer is interpolated from two dilutions that encompass this 50% kill cutoff.

Endpoint titers of 1:8 or greater were considered positive results in these killing type OPAs.

In one embodiment, the immunogenic composition of the invention is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9N in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay (OPA). In one embodiment, the immunogenic composition of the invention is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9N in at least 60%, 70%, 80%, 90%, or at least 93% of subjects as determined by an in vitro opsonophagocytic killing assay (OPA).

In one embodiment, the immunogenic composition of the invention is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9A in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay (OPA). In one embodiment, the immunogenic composition of the invention is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9A in at least 60%, 70%, 80%, 90%, or at least 95% of subjects as determined by an in vitro opsonophagocytic killing assay (OPA).

In one embodiment, the immunogenic composition of the invention is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9L in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay (OPA). In one embodiment, the immunogenic composition of the invention is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9L in at least 60%, 70%, 80%, 90%, or at least 95% of subjects as determined by an in vitro opsonophagocytic killing assay (OPA).

In some embodiments, the subject may have a serotype-specific OPA titer prior to pneumococcal vaccination due to, for example, natural exposure to S. pneumoniae (eg, in the case of an adult subject).

Therefore, a comparison of OPA activity in sera before and after immunization with the immunogenic composition of the invention can be performed and the responses to serotypes 9A, 9L and 9N can be compared to assess the potential increase in responders (see Example 1).

In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) compared to a previously immunized population.

Thus, in one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to S. pneumoniae serotype 9N compared to a previously immunized population.

In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to S. pneumoniae serotype 9A compared to a previously immunized population.

In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to S. pneumoniae serotype 9L compared to a previously immunized population.

In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to S. pneumoniae serotypes 9N and 9A compared to a previously immunized population.

In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to S. pneumoniae serotypes 9N and 9L compared to a previously immunized population.

In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to S. pneumoniae serotypes 9A and 9L compared to a previously immunized population.

In one embodiment, the immunogenic composition of the invention significantly increases the proportion of responders (ie, individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to S. pneumoniae serotypes 9N, 9A, and 9L compared to a previously immunized population.

OPA activity can also be compared before and after immunization with the immunogenic composition of the invention by comparing potential increases in OPA titers.

Therefore, a comparison of OPA activity in sera before and after immunization with the immunogenic composition of the invention and the response to serotypes 9A, 9L and 9N can be performed to evaluate potential increases in OPA titers (see Example 1).

In one embodiment, the immunogenic composition of the invention is capable of significantly increasing OPA titers in a human subject compared to a previously vaccinated population.

Thus, in one embodiment, the immunogenic composition of the invention significantly increases OPA titers against S. pneumoniae serotype 9N in a human subject compared to a previously vaccinated population. In one embodiment, the fold increase in OPA titers against S. pneumoniae serotype 9N is at least 1.2, 1.5, 1.75, 2.0, or 2.1.

In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer against S. pneumoniae serotype 9A in a human subject compared to a previously immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 9A is at least 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, or 7.5.

In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer against S. pneumoniae serotype 9L in a human subject compared to a previously immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 9L is at least 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.2.

In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer against S. pneumoniae serotypes 9N and 9A in a human subject compared to a previously immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 9N is at least 1.2, 1.5, 1.75, 2.0, or 2.1, and the fold increase in OPA titer against S. pneumoniae serotype 9A is at least 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, or 7.5.

In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer against S. pneumoniae serotypes 9N and 9L in a human subject compared to a previously immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 9N is at least 1.2, 1.5, 1.75, 2.0, or 2.1, and the fold increase in OPA titer against S. pneumoniae serotype 9L is at least 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.2.

In one embodiment, the immunogenic composition of the invention significantly increases OPA titers against S. pneumoniae serotypes 9A and 9L in a human subject compared to a previously immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 9A is at least 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, or 7.5, and the fold increase in OPA titer against S. pneumoniae serotype 9L is at least 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.2.

In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer against S. pneumoniae serotypes 9N, 9A, and 9L in a human subject compared to a previously immunized population. In one embodiment, the fold increase in OPA titer against S. pneumoniae serotype 9N is at least 1.2, 1.5, 1.75, 2.0, or 2.1, the fold increase in OPA titer against S. pneumoniae serotype 9A is at least 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, or 7.5, and the fold increase in OPA titer against S. pneumoniae serotype 9L is at least 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.2.

7. Application of the Immunogenic Composition of the Present Invention

In one embodiment, the immunogenic compositions disclosed herein are used as medicaments.

The immunogenic compositions disclosed herein can be used in various therapeutic or prophylactic methods to prevent, treat, or alleviate bacterial infections, diseases, or conditions in a subject. In particular, the immunogenic compositions of the present invention can be used to prevent, treat, or alleviate Streptococcus pneumoniae infections, diseases, or conditions in a subject.

Thus, in one aspect, the present invention provides a method for preventing, treating or ameliorating a S. pneumoniae-associated infection, disease or condition in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the present invention provides a method for preventing, treating or ameliorating an infection, disease or condition associated with S. pneumoniae serotypes 9N, 9A and 9L in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the present invention provides a method for preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotype 9N in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the present invention provides a method for preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotype 9A in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the present invention provides a method for preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotype 9L in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the present invention provides a method for preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotypes 9N and 9A in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the present invention provides a method for preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotypes 9N and 9L in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the present invention provides a method for preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotypes 9A and 9L in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention.

In one aspect, the invention provides a method of inducing an immune response against S. pneumoniae serotypes 9N, 9A and/or 9L in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention.

In one aspect, the invention provides a method of inducing an immune response against S. pneumoniae serotype 9N in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention.

In one aspect, the invention provides a method of inducing an immune response against S. pneumoniae serotype 9A in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention.

In one aspect, the invention provides a method of inducing an immune response against S. pneumoniae serotype 9L in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the invention.

In one aspect, the immunogenic compositions of the invention are used in a method for preventing, treating, or ameliorating an infection, disease, or condition caused by S. pneumoniae serotypes 9N, 9A, and/or 9L in a subject.

In one aspect, the immunogenic compositions of the present invention are used in a method for preventing, treating, or ameliorating an infection, disease, or condition caused by S. pneumoniae serotype 9N in a subject. In one aspect, the immunogenic compositions of the present invention are used in a method for preventing, treating, or ameliorating an infection, disease, or condition caused by S. pneumoniae serotype 9A in a subject. In one aspect, the immunogenic compositions of the present invention are used in a method for preventing, treating, or ameliorating an infection, disease, or condition caused by S. pneumoniae serotype 9L in a subject.

In one aspect, the immunogenic compositions of the invention are used in a method for preventing, treating, or ameliorating an infection, disease, or condition caused by S. pneumoniae serotypes 9N and 9A in a subject. In one aspect, the immunogenic compositions of the invention are used in a method for preventing, treating, or ameliorating an infection, disease, or condition caused by S. pneumoniae serotypes 9N and 9L in a subject. In one aspect, the immunogenic compositions of the invention are used in a method for preventing, treating, or ameliorating an infection, disease, or condition caused by S. pneumoniae serotypes 9A and 9L in a subject.

In one embodiment, any of the immunogenic compositions disclosed herein are used in a method of immunizing a subject against infection by S. pneumoniae serotypes 9N, 9A, and/or 9L.

In one embodiment, any of the immunogenic compositions disclosed herein are used in a method for immunizing a subject against infection with S. pneumoniae serotype 9N. In one embodiment, any of the immunogenic compositions disclosed herein are used in a method for immunizing a subject against infection with S. pneumoniae serotype 9A. In one embodiment, any of the immunogenic compositions disclosed herein are used in a method for immunizing a subject against infection with S. pneumoniae serotype 9L.

In one embodiment, any of the immunogenic compositions disclosed herein are used in a method for immunizing a subject against infection by S. pneumoniae serotypes 9N and 9A. In one embodiment, any of the immunogenic compositions disclosed herein are used in a method for immunizing a subject against infection by S. pneumoniae serotypes 9N and 9L. In one embodiment, any of the immunogenic compositions disclosed herein are used in a method for immunizing a subject against infection by S. pneumoniae serotypes 9A and 9L.

In one aspect, the present invention relates to the use of the immunogenic composition disclosed herein in the manufacture of a medicament for preventing, treating or ameliorating an infection, disease or condition caused by S. pneumoniae serotypes 9N, 9A and/or 9L in a subject.

In one aspect, the present invention relates to the use of the immunogenic compositions disclosed herein in the manufacture of a medicament for preventing, treating, or alleviating an infection, disease, or condition caused by S. pneumoniae serotypes 9N and 9A in a subject. In one aspect, the present invention relates to the use of the immunogenic compositions disclosed herein in the manufacture of a medicament for preventing, treating, or alleviating an infection, disease, or condition caused by S. pneumoniae serotypes 9N and 9L in a subject. In one aspect, the present invention relates to the use of the immunogenic compositions disclosed herein in the manufacture of a medicament for preventing, treating, or alleviating an infection, disease, or condition caused by S. pneumoniae serotypes 9A and 9L in a subject.

In one embodiment, the present invention relates to the use of the immunogenic composition disclosed herein for the manufacture of a medicament for immunizing a subject against infection by S. pneumoniae serotypes 9N, 9A, and/or 9L.

In one embodiment, the present invention relates to the use of an immunogenic composition disclosed herein for the production of a medicament for immunizing a subject against infection with Streptococcus pneumoniae serotype 9N. In one embodiment, the present invention relates to the use of an immunogenic composition disclosed herein for the production of a medicament for immunizing a subject against infection with Streptococcus pneumoniae serotype 9A. In one embodiment, the present invention relates to the use of an immunogenic composition disclosed herein for the production of a medicament for immunizing a subject against infection with Streptococcus pneumoniae serotype 9L.

In one embodiment, the present invention relates to the use of an immunogenic composition disclosed herein for the production of a medicament for immunizing a subject against infection by S. pneumoniae serotypes 9N and 9A. In one embodiment, the present invention relates to the use of an immunogenic composition disclosed herein for the production of a medicament for immunizing a subject against infection by S. pneumoniae serotypes 9N and 9L. In one embodiment, the present invention relates to the use of an immunogenic composition disclosed herein for the production of a medicament for immunizing a subject against infection by S. pneumoniae serotypes 9A and 9L.

In one aspect, the present invention provides a method of inducing an immune response against Streptococcus pneumoniae serotype 9N, 9A and/or 9L in a subject. In one aspect, the present invention provides a method of inducing an immune response against Streptococcus pneumoniae serotype 9N in a subject. In one aspect, the present invention provides a method of inducing an immune response against Streptococcus pneumoniae serotype 9A in a subject. In one aspect, the present invention provides a method of inducing an immune response against Streptococcus pneumoniae serotype 9L in a subject. In one embodiment, the immunogenic compositions disclosed herein are used as vaccines. More particularly, the immunogenic compositions disclosed herein can be used to prevent Streptococcus pneumoniae serotype 9N, 9A and/or 9L infection in a subject. Therefore, in one aspect, the present invention provides a method of preventing Streptococcus pneumoniae serotype 9N, 9A and/or 9L infection in a subject, comprising administering to the subject an immunologically effective amount of the immunogenic composition of the present invention. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural effusion, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess. In one aspect, the subject to be vaccinated is a mammal, such as a human, cat, sheep, pig, horse, cow, or dog.

In one aspect, the immunogenic compositions disclosed herein are used in a method for preventing, treating, or ameliorating an infection, disease, or condition associated with Streptococcus pneumoniae serotypes 9N, 9A, and/or 9L in a subject. In some such embodiments, the infection, disease, or condition is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess.

In one aspect, the immunogenic compositions disclosed herein are used in a method for preventing infection with Streptococcus pneumoniae serotypes 9N, 9A, and/or 9L in a subject. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural effusion, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess. In one aspect, the subject to be vaccinated is a mammal, such as a human, cat, sheep, pig, horse, cattle, or dog.

In one aspect, the present invention relates to the use of the immunogenic compositions disclosed herein for the manufacture of a medicament for preventing, treating, or ameliorating an infection, disease, or condition associated with Streptococcus pneumoniae serotypes 9N, 9A, and/or 9L in a subject. In some such embodiments, the infection, disease, or condition is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess.

In one aspect, the present invention relates to the use of the immunogenic compositions disclosed herein for the manufacture of a medicament for preventing an infection associated with Streptococcus pneumoniae serotypes 9N, 9A, and/or 9L in a subject. In some such embodiments, the infection is selected from the group consisting of pneumonia, sinusitis, otitis media, acute otitis media, meningitis, bacteremia, sepsis, pleural empyema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection, and brain abscess. In one aspect, the subject to be vaccinated is a mammal, such as a human, cat, sheep, pig, horse, cattle, or dog.

The immunogenic compositions of the present invention can be used to protect or treat humans susceptible to infection with S. pneumoniae serotypes 9N, 9A, and/or 9L by administering the immunogenic compositions systemically or via mucosal routes. In one embodiment, the immunogenic compositions disclosed herein are administered intramuscularly, intraperitoneally, intradermally, or subcutaneously. In one embodiment, the immunogenic compositions disclosed herein are administered by intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In one embodiment, the immunogenic compositions disclosed herein are administered by intramuscular or subcutaneous injection.

In one embodiment, the immunogenic composition of the invention comprises at least one saccharide conjugate from S. pneumoniae 9V (such as the saccharide conjugates described in paragraph 1.3 above).

8. Subjects treated with the immunogenic compositions of the present invention

As disclosed herein, the immunogenic compositions described herein can be used in various therapeutic or prophylactic methods to prevent, treat, or ameliorate a bacterial infection, disease, or condition in a subject.

In a preferred embodiment, the subject is a human. In a most preferred embodiment, the subject is a neonate (i.e., under three months of age), an infant (i.e., between three months and one year of age), or a toddler (i.e., between one and four years of age).

In one embodiment, the immunogenic compositions disclosed herein are used as vaccines.

In this embodiment, the age of the subject to be vaccinated can be less than 1 year old. For example, the age of the subject to be vaccinated can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 months old. In one embodiment, the age of the subject to be vaccinated is about 2, 4 or 6 months old. In another embodiment, the age of the subject to be vaccinated is less than 2 years old. For example, the age of the subject to be vaccinated can be about 12 to about 15 months old. In some cases, only one dose of the immunogenic composition of the present invention is required, but in some cases a second, third or fourth dose can be given (see Section 9 below).

In one embodiment of the present invention, the subject to be vaccinated is an adult of 50 years of age or older, preferably an adult of 55 years of age or older. In one embodiment, the subject to be vaccinated is an adult of 65 years of age or older, 70 years of age or older, 75 years of age or older, or 80 years of age or older.

In one embodiment, the subject to be vaccinated is an immunocompromised individual, particularly a human. An immunocompromised individual is generally defined as a person who exhibits a weakened or reduced ability to mount normal humoral or cellular defenses against attack by infectious agents.

In one embodiment of the invention, the immunocompromised subject to be vaccinated has a disease or condition that weakens the immune system and results in an inadequate antibody response to prevent or treat pneumococcal disease.

In one embodiment, the disease is a primary immunodeficiency disease. Preferably, the primary immunodeficiency disease is selected from the group consisting of: combined T-cell and B-cell immunodeficiency, antibody deficiency, well-defined syndrome, immune disorder, phagocytic disorder, innate immune deficiency, autoinflammatory disease, and complement deficiency. In one embodiment, the primary immunodeficiency disease is selected from the group consisting of the diseases described on page 24, line 11 to page 25, line 19 of WO 2010/125480.

In a specific embodiment of the invention, the immunocompromised subject to be vaccinated has a disease selected from the group consisting of HIV infection, acquired immune deficiency syndrome (AIDS), cancer, chronic heart or lung disease, congestive heart failure, diabetes, chronic liver disease, alcoholism, cirrhosis, spinal fluid leak, cardiomyopathy, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), spleen dysfunction (such as sickle cell disease), asplenia (asplenia), hematological malignancies, leukemia, multiple myeloma, Hodgkin's disease, lymphoma, renal failure, nephrotic syndrome and asthma.

In one embodiment of the invention, the immunocompromised subject to be vaccinated suffers from a nutritional disorder.

In a specific embodiment of the present invention, the immunocompromised subject to be vaccinated is currently taking a drug or treatment that reduces the body's resistance to infection. In one embodiment, the drug is selected from the drugs disclosed on page 26, line 33 to page 26, line 4 of WO 2010/125480.

In a specific embodiment of the invention, the immunocompromised subject to be vaccinated is a smoker.

In a specific embodiment of the invention, the white blood cell count of the immunocompromised subject to be vaccinated is less than 5×10 ⁹ cells/L, or less than 4×10 ⁹ cells/L, or less than 3×10 ⁹ cells/L, or less than 2×10 ⁹ cells/L, or less than 1×10 ⁹ cells/L, or less than 0.5×10 ⁹ cells/L, or less than 0.3×10 ⁹ cells/L or less than 0.1×10 ⁹ cells/L.

White blood cell count (WBC): The number of white blood cells (WBCs) in the blood. WBCs are usually measured as part of a CBC (complete blood count). White blood cells are infection-fighting cells in the blood, distinct from the red (oxygen-carrying) blood cells called erythrocytes. There are different types of white blood cells, including neutrophils (polymorphonuclear leukocytes; PMNs), band cells (slightly immature neutrophils), T-type lymphocytes (T cells), B-type lymphocytes (B cells), mononuclear cells (monocytes), eosinophils, and basophils. All types of white blood cells are reflected in the white blood cell count. A normal range for a white blood cell count is typically 4,300-10,800 cells/cubic millimeter of blood. This may also be called the leukocyte count and can be expressed in International Units (IUs) of 4.3-10.8 x ¹⁰⁹ cells/L.

In a specific embodiment of the invention, the immunocompromised subject to be vaccinated has neutropenia. In a specific embodiment of the invention, the neutrophil count of the immunocompromised subject to be vaccinated is less than 2×10 ⁹ cells/L, or less than 1×10 ⁹ cells/L, or less than 0.5×10 ⁹ cells/L, or less than 0.1×10 ⁹ cells/L or less than 0.05×10 ⁹ cells/L.

Low white blood cell count, or "neutropenia," is a condition characterized by abnormally low levels of neutrophils circulating in the blood. Neutrophils are a special type of white blood cell that help prevent and fight infection. The most common reason cancer patients experience neutropenia is as a side effect of chemotherapy. Chemotherapy-induced neutropenia increases a patient's risk of infection and interruption of cancer treatment.

In a specific embodiment of the invention, the immunocompromised subject to be vaccinated has a CD4+ cell count below 500/ ^mm3 , or a CD4+ cell count below 300/ ^mm3 , or a CD4+ cell count below 200/ ^mm3 , or a CD4+ cell count below 100/ ^mm3 , 75/ ^mm3 , or a CD4+ cell count below 50/ ^mm3 .

CD4 cell tests typically report the number of cells per ^mm³ . Normal CD4 counts range from 500 to 1600, and CD8 counts range from 375 to 1100. CD4 counts decrease significantly in people with HIV.

In one embodiment of the present invention, any immunocompromised subject disclosed herein is a human male or a human female.

9 plans

In some cases, only one dose of the immunogenic composition of the invention is required, but in some cases, such as in more severe immunodeficiency disorders, a second, third, or fourth dose may be given. Following the initial vaccination, the subject may receive one or more booster immunizations at appropriate intervals.

In one embodiment, the vaccination schedule of the immunogenic composition of the present invention is a single dose. In a specific embodiment, the single dose schedule is for healthy people who are at least 2 years old.

In one embodiment, the vaccination schedule of the immunogenic composition of the present invention is a multiple-dose schedule. In a specific embodiment, the multiple-dose schedule consists of a series of two doses spaced about 1 month to about 2 months apart. In a specific embodiment, the multiple-dose schedule consists of a series of two doses spaced about 1 month apart, or a series of two doses spaced about 2 months apart.

In another embodiment, the multiple dose schedule consists of a series of 3 doses spaced about 1-2 months apart. In another embodiment, the multiple dose schedule consists of a series of 3 doses spaced about 1 month apart, or a series of 3 doses spaced about 2 months apart.

In another embodiment, the multiple-dose schedule consists of a series of 3 doses spaced about 1 to about 2 months apart, followed by a fourth dose about 10 to about 13 months after the first dose. In another embodiment, the multiple-dose schedule consists of a series of 3 doses spaced about 1 month apart, followed by a fourth dose about 10 to about 13 months after the first dose, or a series of 3 doses spaced about 2 months apart, followed by a fourth dose about 10 to about 13 months after the first dose.

In one embodiment, the multiple dose schedule consists of at least one (eg, 1, 2, or 3 doses) dose at one year of age, followed by at least one toddler dose.

In one embodiment, the multiple dose schedule consists of a series of 2 or 3 doses starting at 2 months of age, spaced about 1 to about 2 months apart (e.g., 28-56 days between doses), followed by a toddler dose at 12-18 months of age. In one embodiment, the multiple dose schedule consists of a series of 3 doses starting at 2 months of age, spaced about 1 to 2 months apart (e.g., 28-56 days between doses), followed by a toddler dose at 12-15 months of age. In another embodiment, the multiple dose schedule consists of a series of 2 doses starting at 2 months of age, spaced about 2 months apart, followed by a toddler dose at 12-18 months of age.

In one embodiment, the multiple dose schedule consists of 4 doses at the ages of 2, 4, 6, and 12-15 months of age.

In one embodiment, a prime dose is administered on day 0, and one or more boosters are administered at dosing intervals of about 2 to about 24 weeks, preferably at dosing intervals of 4 to 8 weeks.

In one embodiment, the initial dose is given on day 0, and the boost is given approximately 3 months later.

10 Kits and Methods

In one embodiment, the present invention relates to a kit comprising the immunogenic composition disclosed herein and an information leaflet.

In one embodiment, the information leaflet refers to the ability of the composition to elicit functional antibodies to S. pneumoniae serotypes 9A, 9L and/or 9N.

In one embodiment, the information leaflet refers to the ability of the composition to elicit functional antibodies to S. pneumoniae serotype 9N.

In one embodiment, the information leaflet refers to the ability of the composition to elicit anti-capsular antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population at a concentration of ≥ 0.35 μg/mL.

In one embodiment, the information leaflet refers to the ability of the composition to elicit anti-capsular antibodies against S. pneumoniae serotype 9N in a human population at a concentration of ≥ 0.35 μg/mL.

In one embodiment, the information leaflet refers to the ability of the composition to elicit OPA titers against S. pneumoniae serotypes 9A, 9L, and/or 9N in a human population.

In one embodiment, the information leaflet refers to the ability of the composition to elicit OPA titers against S. pneumoniae serotype 9N in a human population.

In one embodiment, the present invention relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, said method comprising the steps of:

- producing the immunogenic composition disclosed in the present invention, and

- combining said immunogenic composition with an information leaflet in the same said kit, wherein said information leaflet mentions the ability of said composition to elicit functional antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N.

In one embodiment, the present invention relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, said method comprising the steps of:

- producing the immunogenic composition disclosed in the present invention, and

- combining said immunogenic composition with an information leaflet in the same said kit, wherein said information leaflet mentions the ability of said composition to elicit anti-capsular antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population at a concentration of ≥ 0.35 μg/mL.

In one embodiment, the present invention relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, said method comprising the steps of:

- producing the immunogenic composition disclosed in the present invention, and

- combining said immunogenic composition with an information leaflet in the same said kit, wherein said information leaflet mentions the ability of said composition to elicit OPA titers against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population.

In one embodiment, the present invention relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, said method comprising the steps of:

- producing the immunogenic composition disclosed in the present invention, and

- a printed information leaflet, wherein the information leaflet refers to the ability of the composition to elicit functional antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N

- combining said immunogenic composition and said information leaflet in the same said kit.

In one embodiment, the present invention relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, said method comprising the steps of:

- producing the immunogenic composition disclosed in the present invention, and

- a printed information leaflet, wherein the information leaflet mentions the ability of the composition to elicit anti-capsular antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N at a concentration of ≥ 0.35 μg/mL in humans

- combining said immunogenic composition and said information leaflet in the same said kit.

In one embodiment, the present invention relates to a method for producing a kit comprising an immunogenic composition and an informational package insert, said method comprising the steps of:

- producing the immunogenic composition disclosed in the present invention, and

- a printed information leaflet, wherein the information leaflet refers to the ability of the composition to elicit OPA titers against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population

- Combining the immunogenic composition and the information leaflet in the same kit. 11 Method

In one embodiment, the present invention relates to a method comprising the steps of:

- injecting a subject with an immunologically effective amount of any immunogenic composition defined in the present invention;

- collecting a serum sample from the subject;

- The serum samples were tested for opsonophagocytic killing activity against S. pneumoniae serotype 9N by in vitro opsonophagocytic killing assay (OPA).

In one embodiment, the present invention relates to a method comprising the steps of:

- injecting a subject with an immunologically effective amount of any immunogenic composition defined in the present invention;

- collecting a serum sample from the subject;

- The serum samples were tested for opsonophagocytic killing activity against S. pneumoniae serotype 9A by in vitro opsonophagocytic killing assay (OPA).

In one embodiment, the present invention relates to a method comprising the steps of:

- injecting a subject with an immunologically effective amount of any immunogenic composition defined in the present invention;

- collecting a serum sample from the subject;

- The serum samples were tested for opsonophagocytic killing activity against S. pneumoniae serotype 9L by in vitro opsonophagocytic killing assay (OPA).

In one embodiment, the present invention relates to a method comprising the steps of:

- injecting a subject with an immunologically effective amount of any immunogenic composition defined in the present invention;

- collecting a serum sample from the subject;

- testing the opsonophagocytic killing activity of the serum samples against S. pneumoniae serotypes 9N, 9A and/or 9L by an in vitro opsonophagocytic killing assay (OPA).

Specific embodiments of the invention are set out in the following numbered paragraphs:

1. An immunogenic composition comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 9V for use in a method of immunizing a subject against infection with Streptococcus pneumoniae serotypes 9N, 9A and/or 9L.

2. The immunogenic composition of paragraph 1, wherein the composition does not comprise capsular saccharides from S. pneumoniae serotype 9N.

3. The immunogenic composition of any of paragraphs 1-2, wherein the composition does not comprise capsular saccharides from S. pneumoniae serotype 9A.

4. The immunogenic composition of any of paragraphs 1-3, wherein the composition does not comprise capsular saccharides from S. pneumoniae serotype 9L.

5. The immunogenic composition of paragraph 1, wherein the composition does not comprise capsular saccharides from S. pneumoniae serotypes 9N, 9A and 9L.

6. The immunogenic composition of any of paragraphs 1-5, further comprising at least one saccharide conjugate from S. pneumoniae serotype 4.

7. The immunogenic composition of any of paragraphs 1-6, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 6B.

8. The immunogenic composition of any of paragraphs 1-7, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 14.

9. The immunogenic composition of any of paragraphs 1-8, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 18C.

10. The immunogenic composition of any of paragraphs 1-9, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 19F.

11. The immunogenic composition of any of paragraphs 1-10, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 23F.

12. The immunogenic composition of any of paragraphs 1-5, further comprising a saccharide conjugate from S. pneumoniae serotypes 4, 6B, 14, 18C, 19F and 23F.

13. The immunogenic composition of any of paragraphs 1-12, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 1.

14. The immunogenic composition of any of paragraphs 1-13, further comprising at least one saccharide conjugate from S. pneumoniae serotype 5.

15. The immunogenic composition of any of paragraphs 1-14, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 7F.

16. The immunogenic composition of any of paragraphs 1-15, further comprising a saccharide conjugate from S. pneumoniae serotypes 1, 5, and 7F.

17. The immunogenic composition of any of paragraphs 1-16, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 6A.

18. The immunogenic composition of any of paragraphs 1-17, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 19A.

19. The immunogenic composition of any of paragraphs 1-15, further comprising saccharide conjugates from S. pneumoniae serotypes 6A and 19A.

20. The immunogenic composition of any of paragraphs 1-19, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 3.

21. The immunogenic composition of any of paragraphs 1-20, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 15B.

22. The immunogenic composition of any of paragraphs 1-21, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 22F.

23. The immunogenic composition of any of paragraphs 1-22, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 33F.

24. The immunogenic composition of any of paragraphs 1-23, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 12F.

25. The immunogenic composition of any of paragraphs 1-24, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 10A.

26. The immunogenic composition of any of paragraphs 1-25, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 11A.

27. The immunogenic composition of any of paragraphs 1-26, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 8.

28. The immunogenic composition of any of paragraphs 1-20, further comprising saccharide conjugates from S. pneumoniae serotypes 22F and 33F.

29. The immunogenic composition of any of paragraphs 1-20, further comprising a saccharide conjugate from S. pneumoniae serotypes 15B, 22F and 33F.

30. The immunogenic composition of any of paragraphs 1-23, further comprising a saccharide conjugate from S. pneumoniae serotypes 12F, 10A, 11A, and 8.

31. The immunogenic composition of any of paragraphs 1-30, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 2.

32. The immunogenic composition of any of paragraphs 1-31, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 17F.

33. The immunogenic composition of any of paragraphs 1-32, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 20.

34. The immunogenic composition of any of paragraphs 1-30, further comprising a saccharide conjugate from S. pneumoniae serotypes 2, 17F, and 20.

35. The immunogenic composition of any of paragraphs 1-34, further comprising at least one saccharide conjugate from Streptococcus pneumoniae serotype 15C.

36. The immunogenic composition of any of paragraphs 1-35, which is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24-valent pneumococcal conjugate composition.

37. The immunogenic composition of any of paragraphs 1-35, which is a 14-, 15-, 16-, 17-, 18-, 19-, or 20-valent pneumococcal conjugate composition.

38. The immunogenic composition of any of paragraphs 1-35, which is a 16-valent pneumococcal conjugate composition.

39. The immunogenic composition of any of paragraphs 1-35, which is a 20-valent pneumococcal conjugate composition.

40. The immunogenic composition of any of paragraphs 1-39, wherein the glycoconjugate is solely conjugated to CRM ₁₉₇ .

41. The immunogenic composition of any of paragraphs 1-39, wherein all glycoconjugates are individually conjugated to _CRM197 .

42. The immunogenic composition of any of paragraphs 1-39, wherein the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14 and/or 23F are individually conjugated to PD.

43. The immunogenic composition of any of paragraphs 9-39 or 42, wherein said conjugate is solely conjugated to TT with a saccharide from S. pneumoniae serotype 18C.

44. The immunogenic composition of any of paragraphs 10-39 or 42-43, wherein the saccharide conjugate from S. pneumoniae serotype 19F is conjugated solely to DT.

45. The immunogenic composition of any of paragraphs 1-44, wherein the glycoconjugate is prepared using CDAP chemistry.

46. The immunogenic composition of any of paragraphs 1-44, wherein the glycoconjugate is prepared by a reductive amination reaction.

47. The immunogenic composition of any of paragraphs 1-44, wherein the glycoconjugate from Streptococcus pneumoniae serotype 6A is prepared by a reductive amination reaction.

48. The immunogenic composition of any of paragraphs 1-44 or 47, wherein the glycoconjugate from Streptococcus pneumoniae serotype 19A is prepared by a reductive amination reaction.

49. The immunogenic composition of any of paragraphs 1-44 or 47-48, wherein the saccharide conjugate from Streptococcus pneumoniae serotype 3 is prepared by a reductive amination reaction.

50. The immunogenic composition of any of paragraphs 1-49, wherein the immunogenic composition further comprises antigens from other pathogens.

51. The immunogenic composition of any of paragraphs 1-49, wherein the immunogenic composition further comprises an antigen selected from the group consisting of diphtheria toxoid (D), tetanus toxoid (T), pertussis antigen (P) (typically acellular antigen (Pa)), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis A virus (HAV) antigen, conjugated Haemophilus influenzae type b capsular saccharide (Hib), inactivated poliovirus vaccine (IPV).

52. The immunogenic composition of any of paragraphs 1-51, wherein the immunogenic composition further comprises at least one adjuvant, most preferably any adjuvant disclosed herein.

53. The immunogenic composition of any of paragraphs 1-51, wherein the immunogenic composition further comprises at least one adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

54. The immunogenic composition of any one of paragraphs 1-51, wherein the immunogenic composition comprises 0.1 mg/mL to 1 mg/mL of elemental aluminum in the form of aluminum phosphate as an adjuvant.

55. The immunogenic composition of any of paragraphs 1-54, which is capable of eliciting IgG antibodies that bind to S. pneumoniae serotype 9N polysaccharide in humans at a concentration of ≥ 0.35 μg/mL as determined by ELISA.

56. The immunogenic composition of any of paragraphs 1-55, which is capable of eliciting IgG antibodies that bind to S. pneumoniae serotype 9A polysaccharide in humans at a concentration of ≥ 0.35 μg/mL as determined by ELISA.

57. The immunogenic composition of any of paragraphs 1-56, which is capable of eliciting IgG antibodies that bind to S. pneumoniae serotype 9L polysaccharide at a concentration of ≥0.35 μg/mL in humans as determined by ELISA.

58. The immunogenic composition of any of paragraphs 1-57, which is capable of eliciting functional antibodies capable of killing S. pneumoniae serotypes 9N, 9A and/or 9L in humans as determined by an in vitro opsonophagocytosis assay (OPA).

59. The immunogenic composition of any of paragraphs 1-58, which is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9N in at least 50% of subjects as determined by an in vitro opsonophagocytosis killing assay (OPA).

60. The immunogenic composition of any of paragraphs 1-59, which is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9A in at least 50% of subjects as determined by an in vitro opsonophagocytosis killing assay (OPA).

61. The immunogenic composition of any of paragraphs 1-60, which is capable of eliciting a titer of at least 1:8 against S. pneumoniae serotype 9L in at least 50% of subjects as determined by an in vitro opsonophagocytosis killing assay (OPA).

62. The immunogenic composition of any one of paragraphs 1-61, which significantly increases the proportion of responders against S. pneumoniae serotype 9N compared to a pre-immunized population.

63. The immunogenic composition of any of paragraphs 1-62, which significantly increases the proportion of responders to S. pneumoniae serotype 9A compared to a pre-immunized population.

64. The immunogenic composition of any of paragraphs 1-63, which significantly increases the proportion of responders to S. pneumoniae serotype 9L compared to a pre-immunized population.

65. The immunogenic composition of any of paragraphs 1-64, which is capable of significantly increasing OPA titers against S. pneumoniae serotype 9N in a human subject compared to a previously immunized population.

66. The immunogenic composition of any of paragraphs 1-65, which is capable of significantly increasing OPA titers against S. pneumoniae serotype 9A in a human subject compared to a previously immunized population.

67. The immunogenic composition of any of paragraphs 1-66, which is capable of significantly increasing OPA titers against S. pneumoniae serotype 9L in a human subject compared to a previously immunized population.

68. The immunogenic composition of any of paragraphs 1-67, for use in a method of immunizing a subject against infection by S. pneumoniae serotype 9N.

69. The immunogenic composition of any of paragraphs 1-67, for use in a method of immunizing a subject against infection by S. pneumoniae serotype 9A.

70. The immunogenic composition of any of paragraphs 1-67, for use in a method of immunizing a subject against infection by S. pneumoniae serotype 9L.

71. The immunogenic composition of any of paragraphs 1-67, for use in a method of immunizing a subject against infection by S. pneumoniae serotypes 9N and 9A.

72. The immunogenic composition of any of paragraphs 1-67, for use in a method of immunizing a subject against infection by S. pneumoniae serotypes 9N and 9L.

73. The immunogenic composition of any of paragraphs 1-67, for use in a method of immunizing a subject against infection by S. pneumoniae serotypes 9A and 9L.

74. The immunogenic composition of any of paragraphs 1-67, for use in a method of immunizing a subject against infection by S. pneumoniae serotypes 9N, 9A, and 9L.

75. The immunogenic composition of any of paragraphs 1-67, for use in a method of preventing, treating or ameliorating an infection, disease or condition caused by S. pneumoniae serotypes 9N, 9A and/or 9L in a subject.

76. The immunogenic composition of any of paragraphs 1-67, for use in preventing serotype 9N, 9A and/or 9L S. pneumoniae infection in a subject.

77. The immunogenic composition of any of paragraphs 1-67, for use in a method of protecting or treating a human susceptible to infection with S. pneumoniae serotypes 9N, 9A and/or 9L by administering the immunogenic composition systemically or transmucosally.

78. A method of preventing, treating, or ameliorating an infection, disease, or condition associated with S. pneumoniae serotypes 9N, 9A, and/or 9L in a subject, comprising administering to the subject an immunologically effective amount of the immunogenic composition of any of paragraphs 1-67.

79. A method of preventing infection with S. pneumoniae serotype 9N, 9A, and/or 9L in a subject, comprising administering to the subject an immunologically effective amount of the immunogenic composition of any of paragraphs 1-67.

80. The immunogenic composition of any of paragraphs 1-67, wherein the subject is a human less than 1 year old.

81. The immunogenic composition of any of paragraphs 1-67, wherein the subject is a human less than 2 years old.

82. The immunogenic composition of any of paragraphs 1-67, wherein the subject is an adult 50 years of age or older.

83. The immunogenic composition of any of paragraphs 1-82, for use in a multiple-dose vaccination schedule.

84. A kit comprising the immunogenic composition disclosed herein and an information leaflet.

85. A kit comprising the immunogenic composition of any of paragraphs 1-67 and an information leaflet.

86. The kit of paragraph 85 or 86, wherein the information leaflet refers to the ability of the composition to elicit functional antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N.

87. The kit of paragraph 85 or 86, wherein the information leaflet refers to the ability of the composition to elicit functional antibodies against S. pneumoniae serotype 9N.

88. The kit of paragraph 85 or 86, wherein the information leaflet refers to the ability of the composition to elicit anti-capsular antibodies against S. pneumoniae serotypes 9A, 9L, and/or 9N in a human population at a concentration of ≥ 0.35 μg/mL.

89. The kit of paragraph 85 or 86, wherein the information leaflet refers to the ability of the composition to elicit anti-capsular antibodies against S. pneumoniae serotype 9N in a human population at a concentration of ≥ 0.35 μg/mL.

90. The kit of any of paragraphs 85-89, wherein the information leaflet refers to the ability of the composition to elicit OPA titers against S. pneumoniae serotypes 9A, 9L, and/or 9N in a human.

91. The kit of any of paragraphs 85-89, wherein the information leaflet refers to the ability of the composition to elicit OPA titers against S. pneumoniae serotype 9N in a human.

92. A method of producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of:

- producing the immunogenic composition of any one of paragraphs 1 to 67, and

- combining said immunogenic composition with said information leaflet in the same said kit, wherein said information leaflet mentions the ability of said composition to elicit functional antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N.

93. A method of producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of:

- producing the immunogenic composition of any one of paragraphs 1 to 67, and

- combining said immunogenic composition with said information leaflet in the same said kit, wherein said information leaflet mentions the ability of said composition to elicit anti-capsular antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population at a concentration of ≥ 0.35 μg/mL.

94. A method of producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of:

- producing the immunogenic composition of any one of paragraphs 1 to 67, and

- combining said immunogenic composition with said information leaflet in the same said kit, wherein said information leaflet refers to the ability of said composition to elicit OPA titers against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population.

95. A method of producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of:

- producing the immunogenic composition of any one of paragraphs 1 to 67, and

- a printed information leaflet, wherein the information leaflet refers to the ability of the composition to elicit functional antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N,

- combining said immunogenic composition and said information leaflet in the same said kit.

96. A method of producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of:

- producing the immunogenic composition of any one of paragraphs 1 to 67, and

- a printed information leaflet, wherein the information leaflet refers to the ability of the composition to elicit anti-capsular antibodies against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population at a concentration of ≥ 0.35 μg/mL,

- combining said immunogenic composition and said information leaflet in the same said kit.

97. A method of producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of:

- producing the immunogenic composition of any one of paragraphs 1 to 67,

- a printed information leaflet, wherein said information leaflet refers to the ability of said composition to elicit OPA titers against S. pneumoniae serotypes 9A, 9L and/or 9N in a human population,

- combining said immunogenic composition and said information leaflet in the same said kit.

98. A method comprising the steps of:

- injecting the subject with an immunologically effective amount of an immunogenic composition as defined in any one of paragraphs 1 to 67,

- collecting a serum sample from said subject,

- testing the opsonophagocytic killing activity of the serum samples against S. pneumoniae serotypes 9N, 9A and/or 9L by in vitro opsonophagocytic killing assay (OPA).

99. A method of inducing an immune response against S. pneumoniae serotype 9N, 9A, and/or 9L in a subject, comprising administering to the subject an immunologically effective amount of the immunogenic composition of any of paragraphs 1-67.

100. Use of the immunogenic composition of any of paragraphs 1-67 in the manufacture of a medicament for immunizing a subject against infection by S. pneumoniae serotypes 9N, 9A and/or 9L.

101. Use of the immunogenic composition of any of paragraphs 1-67 in the manufacture of a medicament for preventing, treating or ameliorating an infection, disease or condition caused by Streptococcus pneumoniae serotypes 9N, 9A and/or 9L in a subject.

102. Use of the immunogenic composition of any of paragraphs 1-67 in the manufacture of a medicament for preventing infection by S. pneumoniae serotypes 9N, 9A and/or 9L in a subject.

As used herein, the term "about" refers to within the statistical mean value range, such as a specified concentration range, time frame, molecular weight, temperature or pH. This scope can be within the order of magnitude of the numerical value or range given, typically within 20%, more typically within 10% and even more typically within 5% or 1%. Sometimes this scope can be within the typical experimental error of the standard method for measuring and/or determining a given numerical value or range. The permissible deviation encompassed by the term "about" is determined according to the particular system being studied and can be easily understood by those skilled in the art. When enumerating a range in this specification, each integer within this range is intended as an embodiment of the present invention.

The inventors intend that the terms "comprising," "including," and "containing" may in each instance be optionally replaced by the term "consisting of," respectively.

All references or patent applications cited in this patent specification are incorporated herein by reference.

The present invention is illustrated in the accompanying examples. Unless otherwise described in detail, the following examples are performed using standard techniques well known and conventional to those skilled in the art. The examples are merely illustrative and do not limit the present invention.

Example

Example 1: Evaluation of cross-reactive opsonophagocytic immune responses within S. pneumoniae serogroup 9

The pneumococcal opsonophagocytosis assay (OPA), which measures the killing of S. pneumoniae cells by phagocytic effector cells in the presence of functional antibodies and complement, is considered an important surrogate for assessing the efficacy of pneumococcal vaccines.

Materials and methods

Two randomly selected subsets of immune sera from adults vaccinated with the 13-valent pneumococcal conjugate vaccine (13vPnC) were tested in the OPA assay against serotypes 9V, 9A, 9L, and 9N. Sera were collected from U.S. clinical trials 6115A1-004 (N=59, post-vaccination) and 6115A1-3005 (N=66, matched pre- and post-vaccination), respectively.

Study 6115A1-3005 (ClinicalTrials.gov Identifier: NCT00546572) is a phase 3, randomized, active-controlled, modified double-blind trial evaluating the safety, tolerability, and immunogenicity of 23-valent pneumococcal polysaccharide vaccine (23vPS) compared with 23-valent pneumococcal polysaccharide vaccine (23vPS) in ambulatory individuals 70 years of age and older who received one dose of 23vPS at least 5 years prior to study enrollment (see http://clinicaltrials.gov/ct2/show/NCT00546572, accessed March 31, 2014).

Study 6115A1-004 (ClinicalTrials.gov Identifier: NCT00427895) is a phase 3, randomized, active-controlled, modified double-blind trial evaluating the safety, tolerability, and immunogenicity of a 13-valent pneumococcal conjugate vaccine (13vPnC) compared with a 23-valent pneumococcal polysaccharide vaccine (23vPS) in adults 60 to 64 years of age who had not received 23vPS vaccination, and of 13vPnC in adults 18 to 59 years of age who had not received 23vPS vaccination (see http://clinicaltrials.gov/show/NCT00427895, accessed March 31, 2014).

The 13-valent pneumococcal conjugate vaccine (13vPnC) tested in these studies contained conjugates from pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually conjugated to the diphtheria toxin cross-reactive material 197 (CRM ₁₉₇ ) carrier protein.

The OPA is used to measure functional antibodies against S. pneumoniae serotypes 9V, 9N, 9A, and/or 9L in human serum. Test serum is set up in an assay reaction that measures the ability of capsular polysaccharide-specific immunoglobulins to opsonize and phagocytose bacteria, triggering complement deposition, thereby promoting phagocytosis and killing of bacteria by phagocytes. The OPA titer is defined as the reciprocal dilution that results in a 50% reduction in bacterial count compared to a control well without test serum. The OPA titer is interpolated from two dilutions that encompass this 50% killing cutoff.

The OPA method is based on the method described by Hu et al. (2005) Clin Diagn Lab Immunol 12:287-295. The heat-inactivated serum to be tested is serially diluted 2.5-fold and added to the assay plate along with the target bacteria and incubated with shaking for 30 minutes. Differentiated HL-60 cells (phagocytes) and baby rabbit serum (3-4 weeks old, Arkansas, 12.5% final concentration) are then added to the wells at an effector cell to target ratio of approximately 200:1 and incubated with shaking at 37°C. To terminate the reaction, 80 μL of 0.9% NaCl is added to all wells, mixed, and a 10 μL aliquot is transferred to the wells of an HTS HV filter plate containing 200 μL of water. The liquid is filtered through the plate under vacuum, and 150 μL of culture medium is added to each well and filtered. The filter plates were then incubated overnight at 37°C with 5% _CO2 and then fixed with destaining solution (Bio-Rad Laboratories, Inc., Hercules, CA). The plates were stained with Coomassie blue and destained again. Colonies were imaged and counted on a Cellular Technology Limited (CTL) (Shaker Heights, OH) analyzer. OPA antibody titers were determined as the reciprocal of the lowest serum dilution that resulted in a 50% reduction in bacterial colony counts compared to serum-free bacteria-effector cell-complement control wells.

Statistical analysis: Pearson two-tailed correlation was calculated.

Results - OPA Response in 9V, 9A, 9L and 9N

Cross-functional responses in immune sera from adults vaccinated with 13vPnC against serotypes 9A, 9L, and 9N, as well as homologous functional responses to serotype 9V, were assessed in separate microcolony opsonophagocytosis assays (mcOPA). Two randomly selected subsets of immune sera from adults vaccinated with 13vPnC were tested. Sera were collected from U.S. clinical trials 6115A1-004 (N=59, post-vaccination) and 6115A1-3005 (N=66, matched pre- and post-vaccination).

Subjects in study 6115A1-004 had not previously received any pneumococcal vaccination and received one dose of 13vPnC as part of the study protocol. Immune sera from study 6115A1-004 showed similar percentages of responders for all serotypes, with values of 98.3%, 98.3%, 100%, and 93.2% for serotypes 9V, 9A, 9L, and 9N, respectively ( FIG1 ), supporting the results from 6115A1-3005 ( FIG2 ). Relatively good correlations in OPA titers were observed between serotypes 9V and 9A (Pearson correlation p=0.5456, p<0.0001) or 9L (p=0.7353, p<0.0001), but not 9N (p=0.1217, p<0.3627).

Subjects in study 6115A1-3005 had received one dose of 23vPS at least 5 years prior to study enrollment and one dose of 13vPnC as part of the study regimen. A cohort of matched pre- and post-vaccination sera (N=66) from adults immunized with 13vPnC (Study 6115A1-3005) was evaluated in the OPA for homologous responses to serotype 9V and cross-reactivity of anti-9V antibodies to serotypes 9A, 9L, and 9N. As shown in FIG2 , relatively high immunity (percent responders) was detected in the OPA assay for 9A (84%), 9A (66%), 9L (82%), and 9N (86%), which may be due to their prior 23Vps immunization, which included unconjugated polysaccharides from serotypes 9V and 9N. However, the percentage responders for all four serotypes increased to 95% or higher after vaccination with 13vPnC, which contained only serotype 9V conjugates from serogroup 9. The fold increase in titer values is shown in Table 1 and was similar between serotypes, also suggesting cross-reactivity.

Table 1: Fold increase in OPA titers before and after vaccination, matched, 13vPnC

A more extensive analysis of the OPA titer distribution is shown in the reverse cumulative distribution curves (RCDC) in Figures 3-6. The RCDC shows an increase in serotype-specific immune responses following vaccination for serotypes 9V, 9A, 9L, and to a lesser extent 9N. The correlation of the titer fold increase between each match/sample was analyzed using Pearson's correlation test. Relatively good correlation of titer fold increase was observed between serotypes 9V and 9A (Pearson correlation p = 0.8720, p < 0.0001) or 9N (p = 0.5801, p < 0.0001), but to a lesser extent with 9L (p = 0.1804, p < 0.1640).

in conclusion

Based on these data, the 13vPnC vaccine is likely to provide broader serotype coverage by providing additional protection against serotypes 9A, 9L, and 9N.

Example 2: Further evaluation of cross-reactive opsonophagocytic immune responses within S. pneumoniae serogroup 9

The ability of 13vPnC-induced serotype 9V-specific antibodies to functionally cross-react with bacteria of serotypes 9A, 9L, and 9N (i.e., kill bacteria in OPA of serotypes 9A, 9L, and 9N) was further evaluated in sera from a Phase III clinical study.

Materials and methods

A subset of randomly selected immune sera from adults vaccinated with the 13-valent pneumococcal conjugate vaccine (13vPnC) was tested in the OPA assay against serotypes 9V, 9A, 9L, and 9N. Sera were collected from pre- and post-vaccination sera from the Japanese adult clinical study B1851088 (ClinicalTrials.gov Identifier: NCT01646398).

Study B1851088 was a phase 3, randomized, modified, double-blind, active-controlled trial evaluating the safety, tolerability, and immunogenicity of a 13-valent pneumococcal conjugate vaccine in Japanese adults 65 years of age or older who had not received pneumococcal vaccination (see https://clinicaltrials.gov/ct2/show/NCT01646398, accessed November 30, 2015).

The 13-valent pneumococcal conjugate vaccine (13vPnC) tested in these studies contained conjugates from pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually conjugated to the diphtheria toxin cross-reactive material 197 (CRM ₁₉₇ ) carrier protein.

OPA and statistical analysis were performed as described in Example 1.

Results - OPA responses in serotypes 9V, 9A, 9L, and 9N

Subjects in study B1851088 had not previously received pneumococcal vaccination and received one dose of 13vPnC or 23vPS (23-valent pneumococcal polysaccharide vaccine consisting of a mixture of purified capsular polysaccharides from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F) as part of the study protocol. Cross-functional responses to serotypes 9A, 9L, and 9N in immune sera from Japanese adults immunized with 13vPnC or 23vPS were evaluated in their respective serogroups 9OPA.

A subset of pre- and post-vaccination sera from adults vaccinated with 13vPnC or 23vPS was tested in the OPA assay for serotypes 9A, 9L, and 9N. 9V OPA titers were obtained from reported clinical data. Sera were selected from the Japanese clinical trial B1851088 13vPnC arm (N=91, matched pre- and post-vaccination sera) and 23vPS arm (N=83, matched pre- and post-vaccination sera). Sera were selected from subjects with low or negative pre-vaccination OPA titers for 9V based on reported clinical data. All subjects from B1851088 had no prior exposure to the 23vPS vaccine.

Relatively low baseline serogroup 9OPA titers were observed before vaccination. Following vaccination with 13vPnC or 23vPS, serogroup 9OPA responses increased to high responder rates: 100%, 98%, 92%, and 96% for 9V, 9A, 9L, and 9N, respectively, for 13vPnC-vaccinated subjects and 100%, 88%, 94%, and 100% for 9V, 9A, 9L, and 9N, respectively, for 23vPS-vaccinated subjects (Figures 7 and 8).

13vPnC contains only serotype 9V polysaccharide from serogroup 9, and 23vPS contains polysaccharides from both 9V and 9N. Therefore, the serotype 9N response is homologous after 23vPS vaccination and heterologous (cross-reacting with serotype 9V) after 13vPnC vaccination.

Serogroup 9 OPA GMTs following vaccination were high for all serogroup 9 serotypes regardless of vaccine received (Figure 9).Figures 10-15 show the inverse cumulative distribution curves for serogroup 9 OPA responses to 13vPnC or 23vPS vaccination.

Functional cross-reactive serogroup 9 OPA responses were assessed in serum samples from three pneumococcal vaccine studies (6115A1-004, 6155A1-3005 (Example 1), and B1851088 (Example 2). Surprisingly, the immune response induced by 13vPnC to serotype 9V cross-reacted to varying degrees with all heterologous serotypes within serogroup 9 (9A, 9L, and 9N). Based on these data, the 13vPnC vaccine should provide additional protection against serotypes 9A, 9L, and 9N in addition to the expected protection against serotype 9V.

All publications and patent applications mentioned in this specification are indicative of the levels of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications will occur within the scope of the appended claims.

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Claims (16)

1. An immunogenic composition comprising at least one glycoconjugate from Streptococcus pneumoniae serotype 9V, used in a method of immunizing human subjects against Streptococcus pneumoniae serotypes 9N, 9A and/or 9L infection, wherein said composition does not contain capsular sugars from Streptococcus pneumoniae serotypes 9N, 9A and 9L. 2. The immunogenic composition of claim 1, further comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 4, 6B, 14, 18C, 19F and 23F. 3. An immunogenic composition for use in any one of claims 1-2, further comprising at least one glycoconjugate from Streptococcus pneumoniae serotypes 1, 3, 5, 6A, 7F and 19A. 4. An immunogenic composition for use in any one of claims 1-3, further comprising glycoconjugates derived from Streptococcus pneumoniae serotypes 22F and 33F. 5. An immunogenic composition for use in any one of claims 1-4, wherein the composition is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24-valent Streptococcus pneumoniae conjugate composition. 6. An immunogenic composition for use in any one of claims 1-5, further comprising at least one adjuvant. 7. The immunogenic composition used in any one of claims 1-6, as determined by ELISA, is capable of eliciting at least 0.35 μg/ml of IgG antibodies capable of binding to Streptococcus pneumoniae serotype 9N polysaccharide in humans. 8. An immunogenic composition for use in any one of claims 1-7, as determined by an in vitro opsonization assay (OPA), which in humans can elicit functional antibodies capable of killing Streptococcus pneumoniae serotypes 9N, 9A and/or 9L. 9. The immunogenic composition used in any one of claims 1-8, as determined by an in vitro opsonization phagocytosis assay (OPA), is capable of generating a titer of at least 1:8 against Streptococcus pneumoniae serotype 9N in at least 50% of subjects. 10. The immunogenic composition as defined in any one of claims 1-9, used in a method of immunizing human subjects against infection with Streptococcus pneumoniae serotypes 9N, 9A, and 9L. 11. The immunogenic composition as defined in any one of claims 1-9, for preventing infection of human subjects with Streptococcus pneumoniae serotypes 9N, 9A and/or 9L. 12. Use of the immunogenic composition as defined in any one of claims 1-9 in the production of a medicament for the prevention, treatment or relief of infections, diseases or conditions associated with Streptococcus pneumoniae serotypes 9N, 9A and/or 9L in human subjects. 13. A kit comprising the immunogenic composition as defined in any one of claims 1-9 and an information leaflet, wherein the information leaflet mentions the ability of the composition to elicit functional antibodies against Streptococcus pneumoniae serotypes 9A, 9L and/or 9N. 14. A method for producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the following steps: - To produce the immunogenic composition as defined in any one of claims 1-9; - A printed informational page, wherein the informational page mentions the ability of the composition to elicit functional antibodies against Streptococcus pneumoniae serotypes 9A, 9L and/or 9N; - The immunogenic composition is combined with the information leaflet in the same kit. 15. Use of the immunogenic composition of any one of claims 1-9 in the production of a medicament for inducing an immune response against Streptococcus pneumoniae serotypes 9N, 9A and/or 9L in human subjects. 16. Use of the immunogenic composition of any one of claims 1-9 in the production of a medicament for the prevention of infection with Streptococcus pneumoniae serotypes 9N, 9A and/or 9L in human subjects.