WO2020152706A1

WO2020152706A1 - Multivalent pneumococcal conjugate vaccine compositions

Info

Publication number: WO2020152706A1
Application number: PCT/IN2020/050067
Authority: WO
Inventors: Rajendar Burki; Rajan Sriraman; Ramesh Venkat Matur; Narender Dev MANTENA; Mahima DATLA
Original assignee: Biological E Limited
Priority date: 2019-01-21
Filing date: 2020-01-21
Publication date: 2020-07-30

Abstract

The present invention relates to multivalent pneumococcal polysaccharide-protein conjugates vaccine composition comprising pneumococcal capsular polysaccharide of one or more Streptococcus pneumoniae serotypes conjugated to one or more carrier proteins.

Description

MULTIVALENT PNEUMOCOCCAL CONJUGATE VACCINE COMPOSITIONS FIELD OF INVENTION

BACKGROUND OF INVENTION

Streptococcus pneumoniae (“pneumococcus”) is a gram-positive bacterium that causes invasive diseases, such as pneumonia, bacteremia and meningitis, and diseases associated with colonization, such as acute otitis media (e.g., colonization of middle ear). These pneumococcus- induced diseases result in morbidity and mortality, particularly in persons less than 24 months old and greater than 60 years old. The rate of pneumococcal pneumonia in the U.S. for persons over 60 years of age is estimated to be 3 to 8 per 100,000. In 20% of cases, pneumococcal pneumonia leads to bacteremia and meningitis collectively having a mortality rate close to 30% despite antibiotic treatment.

Pneumococcal vaccines may be administered to prevent infections. Current vaccines include multivalent pneumococcal polysaccharide (comprises pneumococcal polysaccharides from two or more serotypes) and multivalent pneumococcal polysaccharide protein conjugates. The protective efficacy of the pneumococcal polysaccharide vaccine is known to be related to the concentration of antibody generated against a capsular polysaccharide. Pneumococcus cells are encapsulated with a polysaccharide giving rise to more than 90 different pneumococcus serotypes. The capsule is the principal virulence determinant for pneumococci, as it not only protects the cell’s inner surface from complement mediated cell lysis, it is also poorly immunogenic.

Merck’s Pneumovax^®23 is a multivalent pneumococcal polysaccharide vaccine and contains capsular polysaccharides from 23 pneumococcal serotypes including 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. In addition to Pneumovax^®23, the multivalent pneumococcal polysaccharide vaccines that have been licensed so far proved valuable in preventing pneumococcal disease in adults, particularly, the elderly and those at high-risk. However, infants and young children respond poorly to these pneumococcal polysaccharide vaccines.

Prevnar^®-7 is a pneumococcal polysaccharide -protein conjugate vaccine and includes the seven most frequently isolated pneumococcal polysaccharide serotypes (e.g., 4, 6B, 9V, 14, 18C, 19F, and 23F conjugated to CRM197). Since use of Prevnar^®-7 began in the United States in 2000, there has been a significant reduction in invasive pneumococcal disease (IPD) in children. A 13-valent conjugate vaccine Prevenar-13^®, containing thirteen serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM_197, was developed and approved due to the limitations in serotype coverage with Prevnar^®-7 in certain regions of the world.

Synflorix^® is a pneumococcal vaccine that includes ten polysaccharide serotypes wherein polysaccharide from serotypes 1, 4, 5, 6B, 7, 9V, 14, 23F are conjugated to protein D (PD), serotype 18C conjugated to tetanus toxoid (TT) and serotype 19F conjugated to diphtheria toxoid (DT).

Pneumococcal vaccines have been described in additional references. For example, U.S. Patent No. 5,360,897 discloses an immunogenic conjugate composition comprising a reductive amination product of an intact capsular polymer of the bacterial pathogen Streptococcus pneumoniae having at least two carbonyl groups and a bacterial toxin or toxoid, said vaccine comprising a cross-linked conjugate in which there is a direct covalent linkage between the capsular polymer and the toxin or toxoid.

U.S. Patent No. 5,693,326 provides a generalized method for preparing a conjugate vaccine wherein for activating viral, fungal or bacterial polysaccharides, an organic cyanylating agent is used selected from the group l-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate, N-cyanotriethyl-ammonium tetrafluoroborate, and p-nitrophenylcyanate, to form an activated carbohydrate and is subsequently coupled to the protein or carrier protein.

U.S. Patent No. 5,854,416 discloses amino acid and DNA sequences of 37-kDa protein from S. pneumonia known as PsaA (Pneumococcal surface adhesion A).

U.S. Patent No. 7,862,823 discloses a multivalent conjugate vaccine composition comprising pneumococcal capsular polysaccharides with at least two different carrier proteins, such as DT and TT.

U.S. Patent No. 8,192,746 discloses a 15-valent pneumococcal polysaccharide-protein conjugate vaccine composition having capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F conjugated to CRM_197.

U.S. Patent No. 8,808,708 and U.S. Patent No. 8,603,484 describes a 13-valent immunogenic composition consisting polysaccharide-protein conjugates wherein serotypes consist of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, and carrier protein CRM₁₉₇.

U.S. Patent Publication No. 2010/0074922 A1 discloses an immunogenic composition containing 10 or more serotypes wherein 19F capsular saccharide is conjugated to DT, serotype 18C capsular saccharide is conjugated to tetanus toxoid and serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F capsular saccharides are conjugated to Protein D isolated from Haemophilus influenzae.

U.S. Patent Publication No. 2010/0239604 describes an immunogenic composition comprising multivalent Streptococcus pneumoniae capsular saccharide conjugates from serotypes 19A and 19F wherein serotype 19A is conjugated to a first bacterial toxoid and 19F is conjugated to a second bacterial toxoid and 2-9 of the Streptococcus pneumoniae capsular saccharides are conjugated to protein D.

U.S. Patent Publication No. 2012/321658 A1 discloses an immunogenic composition wherein serotypes 1, 3, 19A and 19F linked to protein carrier(s) either directly or indirectly through a chemistry other than reductive amination, and one or more different saccharides is/are selected from a second group consisting of serotypes 4, 5, 6A, 6B, 7F, 9V, 14, 18C and 23F which is/are linked to a protein carrier(s) by reductive amination.

IN 140/DEL/2011 describes a Streptococcus pneumonia vaccine comprising either of (a) 7 or more (b) 10 or more polysaccharides from different serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising DT, diphtheria toxoid, CRM197, and tetanus toxoid.

WO Publication No. 2013/191459 A1 discloses a conjugated 15 valent composition comprising different serotypes of Streptococcus pneumoniae derived from a capsular polysaccharide 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM197.

WO Publication No. 2014/092377 A1 discloses a 13 valent composition wherein 12 serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F and the last serotype is either 2 or 9N conjugated to CRM197.

WO Publication No. 2014/092378 A1 describes an immunogenic conjugate composition where 12 serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F and remaining one from 22F or 33F conjugated to CRM197.

WO Publication No. 2016/207905 A2 discloses a multivalent Pneumococcal conjugate vaccine (PCV) composition comprising: 1) at least 12 capsular polysaccharides selected from serotypes 1, 3, 4, 5, 6B, 7F, 9N, 9V, 15B, 14, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae activated with CDAP and conjugated to carrier protein CRM197, and 2) a pharmaceutically acceptable carrier, wherein the composition does not contain capsular polysaccharide from serotype 6A. Chinese Patent Application Publication No. CN 101590224 describes a 14 valent pneumococcal polysaccharide-protein conjugate vaccine containing serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM19₇.

Chinese Patent Application Publication No. CN 103623401 discloses a 14 multivalent pneumococcal capsular polysaccharide -protein conjugate composition wherein said 14 different serotype are 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197.

Chinese Patent Application Publication No. CN 103656631 provides a multivalent pneumococcus capsular polysaccharide-protein conjugate composition prepared from capsular polysaccharides of pneumococcus of 24 different serotypes and a carrier protein in a covalent linkage manner, wherein the 24 different serotypes are 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F conjugated to CRM19₇.

Chinese Patent Application Publication No. CN 103656632 discloses a multivalent pneumococcal capsular polysaccharide composition containing serotype 6A and at least one extra serotype selected from the group consisting of 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F conjugated to CRM19₇.

Chinese Patent Application Publication No. CN 104069488 discloses a multivalent pneumococcus capsular polysaccharides of 14 different serotypes and carrier protein, wherein the 14 serotypes include 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197.

Anderson P et al, (2003, Vaccine; 21 (13-14): 1554-9) discloses a comparative study of tetravalent conjugate vaccines with each polysaccharide types 6A, 14, 19F, and 23F separately coupled to tetanus toxoid or diphtheria CRM 197 or a mixture of halved doses of polysaccharide types 6A, 14, 19F, and 23F separately coupled to tetanus toxoid and diphtheria CRM197.

Nurkka et al. (2004, Ped. Inf. Dis. J., 23: 1008-1014) discloses a study of the immunogenicity and safety of an 11 -valent pneumococcal protein D conjugate vaccine where no priming effect was observed for serotype 3 in infants who had received three doses of the vaccine followed by a booster dose of either the same vaccine or a pneumococcal polysaccharide vaccine.

The above-mentioned references disclose, amongst other compositions, methods, and the like, multivalent pneumococcal conjugate vaccines comprising polysaccharides from one or more serotypes as well as conjugation of these polysaccharides with carrier proteins. In view of the different serotypes that are prevalent across various geographical regions, there is a need for additional multivalent pneumococcal vaccine comprising novel conjugates of polysaccharide serotypes with improved immune response, as well as simple and efficient production thereof. Surprisingly, the multivalent pneumococcal conjugate vaccine composition of the present invention offer an improved immune response over the naive multivalent pneumococcal vaccines and existing pneumococcal conjugate vaccines.

SUMMARY OF INVENTION

The present invention provides a 20 valent pneumococcal conjugate vaccine composition, comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B.

In an embodiment, the present invention also provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharide from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B wherein the carrier protein is selected from CRM197 or combination of CRM197 and PsaA or combination of CRM197 and Tetanus toxoid or combination of PsaA and Tetanus toxoid or combination of CRM197, PsaA and Tetanus toxoid.

In an embodiment, the present invention provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B wherein the carrier protein is CRM197.

In an embodiment, the present invention provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B wherein the carrier proteins are CRM197 and PsaA.

In an embodiment, the present invention also provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B wherein at least 13 polysaccharides are conjugated to first carrier protein, at least 3 polysaccharides are conjugated to second carrier protein and at least one carrier protein is conjugated to third carrier protein, wherein the first, second and third carrier protein is CRM₁₉₇, PsaA and Tetanus toxoid. In an embodiment, the present invention also provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B wherein at least 13 polysaccharides are conjugated to first carrier protein, at least 3 polysaccharides are conjugated to second carrier protein, wherein the first and second carrier protein is CRM₁₉₇ and Tetanus toxoid.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 3 and (b) serotype 6B with PsaA.

Figure 2: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 6A-CRM₁₉₇ and (b) serotype 6A with PsaA.

Figure 3: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 6C-CRM₁₉₇ and (b) serotype 6C-PsaA.

Figure 4: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 15A-CRMi₉₇ and (b) serotype 15A-PsaA.

Figure 5: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 15C-CRMi₉₇ and (b) serotype 15C-PsaA.

Figure 6: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 23A-CRMi₉₇ and (b) serotype 23A-PsaA.

Figure 7: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 23B-CRMi₉₇ and (b) serotype 23B-PsaA.

Figure 8: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 24F-CRMi₉₇ and (b) serotype 24F-PsaA.

Figure 9: SEC-HPLC chromatogram illustrating conjugation reaction kinetics of (a) serotype 35B-CRMi₉₇ and (b) serotype 35B-PsaA.

Figure 10: IgG titer values of animals vaccinated animals at pre-immunization stage and after immunization with Formulation of Example 2.

Figure 11: Calculation of the Geometric Mean Fold Rise (GMFR) of different serotypes in F024 immunized rabbits with Formulation of Example 2.

DEFINITIONS

Throughout this invention, the singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, unless the word "or" is expressly limited to mean only a single item exclusive from the other items in reference to a list of two or more items, then the use of "or" in such a list is to be interpreted as including (a) any single item in the list, (b) all of the items in the list, or (c) any combination of the items in the list. Additionally, the terms "comprising" and the like are used throughout this invention to mean including at least the recited feature(s) such that any greater number of the same feature(s) and/or one or more additional types of features are not precluded. Reference herein to "one embodiment," "an embodiment," or similar formulations means that a particular feature of a composition, a composition, a method, or a characteristic described in connection with the embodiment may be included in at least one embodiment of the present technology. Thus, the appearances of such phrases or formulations herein are not necessarily all referring to the same embodiment. Furthermore, various particular features, compositions, methods, or characteristics may be combined in any suitable manner in one or more embodiments.

This invention is not intended to be exhaustive or to limit the present technology to the precise forms disclosed herein. Although specific embodiments are disclosed herein for illustrative purposes, various equivalent modifications are possible without deviating from the present technology, as those of ordinary skill in the relevant art will recognize. In some cases, well-known structures and functions have not been shown and/or described in detail to avoid unnecessarily obscuring the description of the embodiments of the present technology. Although steps of methods may be presented herein in a particular order, in alternative embodiments the steps may have another suitable order. Similarly, certain embodiments of the present technology disclosed in the context of particular embodiments may be combined or eliminated in other embodiments. Furthermore, while advantages associated with certain embodiments may have been disclosed in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages or other advantages disclosed herein to fall within the scope of the present technology. Accordingly, this disclosure and associated technology may encompass other embodiments not expressly shown and/or described herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any immunogenic compositions, vaccine compositions or methods similar or equivalent to those described herein can also be used in the practice or testing of the embodiments of the present invention, representative illustrative methods and compositions are now described. Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods, compositions and combinations.

As used herein, the term“capsular polysaccharide” refers to a layer of polysaccharide external to but contiguous with the cell wall of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B.

As used herein, the terms“immunogenic composition” and“vaccine composition” are used interchangeably.

As used herein, the term“carrier protein” refers to any protein which is coupled or attached or conjugated to polysaccharide, typically for the purpose of enhancing or facilitating detection of the antigen by the immune system. Examples of carrier proteins include, but are not limited to CRM197, PsaA and Tetanus toxoid.

The term“conjugate” or“conjugated” as used herein is used to mean that a Streptococcus pneumoniae capsular polysaccharide is covalently bonded to a carrier protein.

As used herein, the term“adjuvant” refers to the non-antigenic component of the vaccine that enhances the immune response of the antigens of the vaccine by facilitating the contact between the antigen and the immune system by influencing the type and the quality of the immune response generated against an antigen. The adjuvant causes prolonged immune responses against the antigens and also may serve to decrease toxicity of certain antigens or provide solubility to certain antigens.

As used herein, the term“pharmaceutically acceptable carrier(s)” refers to one or more optional components which may be added to the vaccine formulation for administration of the antigens and/or viruses which does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. The term includes one or more excipient, stabilizer, diluents, buffers or surfactants, lyophilization excipient or a combination thereof. By pharmaceutically acceptable or pharmacologically acceptable is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a 20 valent multivalent pneumococcal conjugate vaccine composition.

In an embodiment, the present invention provides a pneumococcal conjugate vaccine composition comprising pneumococcal polysaccharides where one or more of the pneumococcal polysaccharides are native pneumococcal polysaccharides.

In another embodiment, the present invention provides a pneumococcal conjugate vaccine composition comprising pneumococcal polysaccharides where one or more of the pneumococcal polysaccharides are fragmented, each fragmented pneumococcal polysaccharide having an average molecular weight less than that of a native pneumococcal polysaccharide.

In another embodiment, the present invention provides a pneumococcal conjugate vaccine composition comprising pneumococcal polysaccharides where one or more of the pneumococcal polysaccharides are directly coupled to an amino group of the carrier protein or are coupled to the amino group by a spacer, wherein the spacer is selected from the group consisting of cystamine, cysteamine, hexane diamine, adipic acid dihydrazide (ADH), ED AC or EDC and the like.

CRM 197 is a variant of diphtheria toxin and is by itself non-toxic (i.e., toxoid). CRM197 is isolated from cultures of Corynebacterium diphtheriae strain C7 (b 197) grown in casamino acids and yeast extract-based medium. CRM197 may be prepared recombinantly in accordance with the methods described in U.S. Pat. No. 5,614,382. Alternatively, CRM197 is prepared recombinantly in accordance with the methods known in the literature or according to the method disclosed in our PCT publication WO 2016/079755. CRM197 may be purified by ultrafiltration, ammonium sulphate precipitation, and ion-exchange chromatography, methods well known in art.

The present invention provides a pneumococcal conjugate vaccine composition comprising one or more pneumococcal polysaccharides of serotypes conjugated to PsaA carrier protein, wherein the PsaA carrier protein is a modified PsaA and does not include wild-type hydrophobic N-terminal leader peptide and includes 290 amino acids. Carrier proteins are non-toxic and non-reactogenic proteins that are obtainable in a sufficient amount and purity. In some embodiments, the present invention provides a pneumococcal conjugate vaccine composition comprising one or more carrier proteins conjugated to one or more Streptococcus pneumoniae polysaccharides (also referred to herein as“pneumococcal polysaccharides”). By conjugating a pneumococcal polysaccharide to a carrier protein, the pneumococcal polysaccharide has increased immunogenicity over the unconjugated pneumococcal polysaccharide.

The pneumococcal conjugate vaccine composition comprising pneumococcal capsular polysaccharide serotypes each individually conjugated to a carrier protein, referred to herein as polysaccharide-protein conjugates and/or conjugates. When included in the pneumococcal vaccine composition described herein, pneumococcal vaccine is a multivalent pneumococcal polysaccharide-protein conjugate vaccine (also referred to herein as“multivalent conjugate vaccine”,“conjugate vaccine”, and/or“polysaccharide -protein conjugate vaccine”). In addition to the multivalent conjugate vaccine, the present invention provides a process for preparing and/or administering the same to a subject in need thereof.

In one embodiment, the pneumococcal conjugate vaccine composition is a 20 valent immunogenic composition comprising capsular polysaccharide from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C/6D, 15A, 15C, 23A, 23B, 24F and 35B wherein the carrier protein is CRM197 or combination of CRM197, PsaA or CRM197, PsaA and Tetanus toxoid.

In some embodiments of the present invention, a combination of the carrier protein is used, which includes two or more carrier proteins, such as PsaA, CRM197 and tetanus toxoid (TT). For example, some polysaccharides are conjugated to CRM197 as carrier protein and some polysaccharides are conjugated to PsaA or TT and the like.

In some embodiments, the present invention provides a pneumococcal polysaccharide- protein conjugate vaccine composition comprising pneumococcal polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C/6D, 15A, 15C, 23A, 23B, 24F and 35B. The selected pneumococcal polysaccharides are each conjugated individually to carrier protein CRM 197.

In some embodiments, the present invention provides a pneumococcal polysaccharide- protein conjugate vaccine composition comprising pneumococcal polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C/6D, 15A, 15C, 23A, 23B, 24F and 35B. The selected pneumococcal polysaccharides are each individually conjugated to carrier protein wherein a first portion of the selected pneumococcal polysaccharides are conjugated to CRM197 and a second portion of the selected pneumococcal polysaccharides are conjugated to PsaA.

In an embodiment, the present invention provides a 20 valent pneumococcal conjugate vaccine composition, the composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B wherein at least 13 polysaccharides are conjugated to first carrier protein, at least 3 polysaccharides are conjugated to second carrier protein and at least one polysaccharide is conjugated to third carrier protein, wherein the first, second and third carrier protein is CRM197, PsaA and Tetanus toxoid respectively.

In some embodiments, the present invention provides a pneumococcal polysaccharide- protein conjugate vaccine composition comprising pneumococcal polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C/6D, 15A, 15C, 23A, 23B, 24F and 35B . The selected pneumococcal polysaccharides are each individually conjugated to carrier protein wherein at least 3 polysaccharides are conjugated to TT, a portion of the selected pneumococcal polysaccharides are conjugated to CRM197 and another portion of the selected pneumococcal polysaccharides are conjugated to PsaA.

In some embodiments, the pneumococcal polysaccharides useful in the compositions of the present invention may be extracted from one or more microorganisms (e.g. Streptococcus pneumoniae ) according to conventional methods. For example, pneumococcal polysaccharides may be prepared according to known procedures. Furthermore, purification of the pneumococcal polysaccharides may be performed according to the procedure described in PCT publication WO 2016/174683 Al.

The extracted pneumococcal polysaccharides may be purified according to conventional methods and may be used in its native form. In other embodiments, the extracted and purified pneumococcal polysaccharides may be fragmented to obtain one or more portions of the pneumococcal polysaccharide, each portion of the pneumococcal polysaccharide having an average molecular weight less than that of the extracted and purified pneumococcal polysaccharides.

In another embodiment, the present invention provides a pneumococcal conjugate vaccine composition comprising pneumococcal polysaccharides, each pneumococcal polysaccharide having an average molecular weight between about 150 kDa and 2000 kDa. In another embodiment, the present invention provides a pneumococcal conjugate vaccine composition comprising one or more pneumococcal capsular polysaccharide serotypes each individually conjugated to a carrier protein, such as a polysaccharide-protein conjugate wherein each polysaccharide-protein conjugate has a molecular weight of about 1,500 kDa to about 15,000 kDa.

In other embodiments, the extracted and purified pneumococcal polysaccharides may be activated prior to conjugation to one or more carrier proteins. For example, the extracted and purified pneumococcal polysaccharides may be activated (e.g., chemically) prior to conjugation to one or more carrier proteins. In other embodiments, one or more of the activated pneumococcal polysaccharides may be conjugated to an individual carrier protein. The conjugates may be prepared by known techniques.

In some embodiments, the pneumococcal polysaccharides may be chemically activated and subsequently conjugated to carrier proteins according to known techniques, such as those described in U.S. Pat. Nos. 4,365,170, 4,673,574 and 4,902,506. For example, pneumococcal polysaccharides can be activated by oxidation of a terminal hydroxyl group to an aldehyde with an oxidizing agent, such as periodate (e.g., sodium periodate, potassium periodate, or periodic acid) by random oxidative cleavage of one or more vicinal hydroxyl groups of the carbohydrates and formation of one or more reactive aldehyde groups.

The pneumococcal polysaccharides may also be activated by CDAP (l-cyano-4- dimethylamino-pyridinium tetrafluoroborate) and subsequently conjugated to one or more carrier proteins such as PsaA, CRM197_, PspA, or combination thereof. In other embodiments, pneumococcal polysaccharides activated with CDAP to form a cyanate ester may be directly conjugated to one or more carrier proteins or conjugated using a spacer (e.g., linker). The spacer may couple to an amino group on the carrier protein. In some embodiments, the spacer may be cystamine or cysteamine, which generates a thiolated polysaccharide that may be coupled to the carrier protein through a thioether linkage to a maleimide-activated carrier protein (e.g., using GMBS) or a haloacetylated carrier protein (e.g., using iodoacetimide, ethyl iodoacetimide HC1, SIAB, SIA, SBAP, and/or N-succinimidyl bromoacetate. In other embodiments, the cyanate ester is coupled using hexane diamine or adipic acid dihydrazide (ADH) and an amino- derivitized saccharide is conjugated to a carrier protein using carbodiimide (e.g. ED AC or EDC) chemistry via a carboxyl group on the protein carrier. Such conjugates are described in PCT Publication No. WO 93/15760, PCT Publication No. WO 95/08348, PCT Publication No. WO 96/29094, and Chu et ah, 1983, Infect. Immunity 40:245-256. Other suitable activation and/or coupling techniques for use with the polysaccharide-protein conjugates and vaccine compositions of the present invention include use of carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S— NHS, EDC, TSTU, and other methods described in PCT Publication No. WO 98/42721. For example, conjugation may involve a carbonyl linker which may be formed by reaction of a free hydroxyl group of the saccharide with CDI (See Bethell et al., 1979, J. Biol. Chem. 254:2572-4; Hearn et al., 1981, J. Chromatogr. 218:509-18) followed by coupling with a protein to form a carbamate linkage. In some embodiments, the anomeric terminus may be reduced to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate and coupling the CDI carbamate intermediate with an amino group on a protein. For example, another suitable activation and/or coupling techniques for use with the polysaccharide-protein conjugates and vaccine compositions of the present invention include the following method: sized pneumococcal polysaccharides (e.g., about 6 mF of sized polysaccharide at a concentration of about 10 mg/mF) and CDAP (e.g., about 100 mg/mF in acetonitrile (w/v)) can be mixed in a glass vial in a ratio of about 1 to about 1 (e.g., by stirring for about 1 minute). The pH of the polysaccharide solution may be adjusted as necessary (e.g., to about 9.25 with about 0.2M triethylamine and stirred for 3 min at room temperature). In addition, PsaA (e.g., about 4 mF of a solution having a concentration of about 15 mg/mF) may be added slowly to the activated pneumococcal polysaccharides (e.g., in a ratio of about 1 to about 1 (Ps: Carrier protein)). The pH of the reaction may be adjusted (e.g., to about 9.05 using 0.2M trimethylamine) and the reaction may be continued (e.g., by stirring for 5 hours at room temperature). The reaction mixture may be quenched (e.g., by addition of an excess concentration of glycine). The reaction mixture may be diafiltered using a membrane (e.g., a 100 K MWCO membrane) and may be purified by size- exclusion chromatography. The diafiltered and purified fractions may be analyzed using SEC- MAFFS, and an anthrone method. The analyzed fractions containing conjugates may be pooled and sterile filtered (e.g., using 0.2 pm filters).

Following conjugation of pneumococcal polysaccharides to one or more carrier proteins, the polysaccharide-protein conjugates may be purified (e.g., enriched with respect to the amount of poly saccharide -protein conjugate) by a variety of techniques. These techniques include, but are not limited to concentration/diafiltration operations, precipitation/elution, column chromatography, and depth filtration. For example, after the conjugates are purified, the conjugates may be compounded to formulate the pneumococcal polysaccharide-protein conjugate compositions of the present invention, which may be used as vaccines.

The pneumococcal polysaccharide -protein conjugate compositions of the present invention further comprise one or more of the following: a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a buffer, a preservative, a stabilizer, an adjuvant, and/or a lyophilization excipient. For example, the pneumococcal polysaccharide-protein conjugate compositions of the present invention may comprise a pharmaceutically acceptable carrier.

In some embodiments, the present invention provides a method for preparing a polysaccharide-protein conjugate of the pneumococcal vaccine composition described herein wherein the method further comprises formulating the polysaccharide-protein conjugate into the pneumococcal vaccine composition including an adjuvant, an excipient, and a buffer.

In some embodiments, the present invention provides a method for preparing a polysaccharide-protein conjugate of the pneumococcal vaccine composition described herein wherein the adjuvant is aluminum phosphate.

In some embodiments, the present invention provides a method of treating a subject in need thereof comprising, administering a pneumococcal vaccine composition described herein to the subject in need thereof.

In some embodiments, the subject has a disease mediated by Streptococcus pneumoniae, such as invasive pneumococcal disease (IPD).

In one embodiment, the subject is a human, such as an infant (less than about 1 year of age), a toddler (about 12 months to about 24 months of age), a young child (about 2 years to about 5 years of age), an older child (about 5 years to about 13 years of age), an adolescent (about 13 years to about 18 years of age), an adult (about 18 years to about 65 years of age), or an elder (more than about 65 years of age).

In some embodiments, the present disclosure provides a method of inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate comprising administering an immunologically effective amount of the pneumococcal conjugate vaccine composition described herein to a subject.

In one embodiment, method of inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising administering the pneumococcal conjugate vaccine composition described herein to the subject systemically, subcutaneously, and/or mucousally. In some embodiments, an amount of each conjugate in a dose of the vaccine composition of the present invention is an amount sufficient to induce an immunoprotective response, such as an immunoprotective response without significant, adverse effects. While the amount of each conjugate may vary depending upon the pneumococcal serotype, each dose of the vaccine compositions may comprise about 0.1 pg to about 50 pg of each pneumococcal polysaccharide, about 0.1 pg to about 10 pg, or about 1 pg to about 5 pg of each pneumococcal polysaccharide conjugated to each carrier protein comprising about 1.5 pg to about 5 pg of carrier protein.

In another embodiment, the present invention provides a pneumococcal conjugate vaccine composition comprising pneumococcal polysaccharides and carrier proteins, the pneumococcal conjugate vaccine composition having a percent ratio of protein to polysaccharide (protein/PS) of about 0.3 to about 2.0 protein/PS, preferably, 0.5 to 1.5.

In some embodiments, the purified polysaccharides before conjugation have a molecular weight of between 10 kDa and 2,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 50 kDa and 2,000 kDa; between 50 kDa and 2,000 kDa; between 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; b 100 kDa and 2,000 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa.

In other embodiments, the present invention provides pneumococcal polysaccharide- protein conjugate vaccine compositions comprising one or more polysaccharide-protein conjugates having a molecular weight ranging between about 1,000 kDa to about 10,000 kDa, about 1,500 kDa to about 15,000 kDa, about 2,000 kDa to about 20,000 kDa, about 2,500 kDa to about 25,000 kDa, or about 3,000 kDa to about 30,000 kDa.

In yet another embodiment, the present invention provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B and the carrier protein is CRM197, PsaA and Tetanus toxoid, wherein each polysaccharide conjugate having a molecular weight ranging between about 1,000 kDa to about 30,000 kDa and the ratio of protein to polysaccharide (protein/PS) of about 0.3 to about 2.0 protein/PS, preferably, 0.5 to 1.5. In a preferred embodiment, the present invention provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B and the carrier protein is CRM197, PsaA and Tetanus toxoid, wherein each polysaccharide conjugate having a molecular weight ranging between about 1,000 kDa to about 15,000 kDa and the ratio of protein to polysaccharide (protein/PS) of 0.5 to 1.5.

In yet another preferred embodiment, the present invention provides a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes comprise 1, 3, 4, 5, 6A, 6B, 6C/6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B and the carrier protein is a combination of CRM 197 and PsaA, wherein each polysaccharide conjugate having a molecular weight ranging between about 1,000 kDa to about 15,000 kDa and the ratio of protein to polysaccharide (protein/PS) of 0.5 to 1.5.

The pneumococcal polysaccharide-protein conjugate vaccine compositions of the present invention may be manufactured using known methods. For example, the pneumococcal polysaccharide-protein conjugate vaccine compositions may be formulated with a pharmaceutically acceptable diluent or vehicle, e.g. water or a saline solution. In addition, the pneumococcal polysaccharide -protein conjugate vaccine compositions may further include one or more of the following: a buffer, a preservative or a stabilizer, polysorbate, an adjuvant such as an aluminum compound, e.g. an aluminium hydroxide, an aluminium phosphate or an aluminium hydroxyphosphate, and/or a lyophilization excipient. Inclusion of any one of the above compounds in the pneumococcal polysaccharide-protein conjugate vaccine compositions of the present invention may be selected as a function of the mode and route of administration to a subject in need thereof and may further be based on standard pharmaceutical practices.

In some embodiments, the present invention provides a method for preparing a 20 valent pneumococcal polysaccharide -protein conjugate composition comprising pneumococcal polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C/6D, 15A, 15C, 23A, 23B, 24F and 35B wherein the carrier protein is CRM197. The method for preparing the 20 valent pneumococcal polysaccharide-protein conjugate composition comprises the steps of;

(a) individually conjugating one or more of the twenty pneumococcal polysaccharides (e.g., activated utilizing CDAP) to CRM197 carrier protein, (b) diafiltering and purifying the conjugates using size exclusion chromatography ,

(c) analyzing the purified fractions using SEC-MALLS, pooling fractions containing each of the twenty conjugates, and filter sterilizing the monovalent conjugate fractions, and

(d) formulating the 20 conjugates (e.g., about 2.2 to 4.4 pg for each serotype, about 30 pg to about 50 pg of CRM197), an adjuvant (such as aluminum phosphate), an excipient, and buffer to provide 20 valent pneumococcal polysaccharide-protein conjugate composition.

In some embodiments, the 20-valent pneumococcal polysaccharide -protein conjugate composition may be filtered (e.g., aseptically).

In some embodiments, the present invention provides a method for preparing a twenty valent pneumococcal polysaccharide -protein conjugate composition comprising pneumococcal polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7L, 9V, 11A, 12L, 14, 15B, 18C, 19A, 19L, 22L, 23L, 33L, 35B and 45 wherein at least serotypes 3, 6A and 6B are conjugated to PsaA and one or more serotypes 1, 4, 5, 7L, 9V, 11A, 12L, 14, 15B, 18C, 19A, 19L, 22L, 23L, 33L, 35B and 45 are conjugated to CRM197. The method for preparing the twenty valent pneumococcal polysaccharide-protein conjugate composition comprises the steps of;

(a) individually conjugating one or more of the twenty pneumococcal polysaccharides (e.g., activated utilizing CDAP) to an immunogenic carrier protein, such as PsaA and/or CRM197,

(b) diafiltering and purifying the conjugates using size exclusion chromatography ,

(d) formulating the twenty conjugates (e.g., about 2.2 to 4.4 pg for each serotype, about 5 pg to about 20 pg of PsaA, and about 20 pg to about 50 pg of CRM197), an adjuvant (such as aluminum phosphate), an excipient, and buffer to provide twenty valent pneumococcal polysaccharide-protein conjugate composition.

The compositions of the present invention may be formulated into a unit dose vial, multiple dose vial, or a pre-filled syringe. The compositions of the present invention may further comprise of one or more preservative(s) selected from thiomersal, 2-phenoxyethanol, m-cresol, benzyl alcohol, benzoic acid and the like or mixture thereof, in an amount which may range from about 2 to 14 mg/mL.

In some embodiments, the present invention also provides an immunogenic composition (e.g., a vaccine), such as a pneumococcal polysaccharide-protein conjugate composition, administered as a single dose of about 0.5 mL formulated to contain at least the following: about 2.2 to 4.4 pg of each of pneumococcal polysaccharide serotypes, about 1 pg to about 10 pg of PsaA per serotype, about 10 pg to about 40 pg of CRM197 for each serotype, about 0.2 mg to about 1 mg of an adjuvant (e.g., aluminum phosphate), and one or more excipients (e.g., sodium chloride, and/or a buffer).

Compositions of the present invention may be administered to a subject in need thereof by any number of conventional routes used in the field of vaccines. For example, compositions of the present invention may be administered systemically, such as parenterally (e.g. subcutaneously, intramuscularly, intradermally and/or intravenously) or mucosally (e.g., orally and/or nasally).

In some embodiments, the present invention also provides methods of inducing an immune response in a subject in need thereof to one or more Streptococcus pneumoniae capsular polysaccharides conjugated to one or more carrier proteins. The methods for inducing the immune response comprise administering an immunologically effective amount of the compositions described herein to the subject in need thereof.

As used herein, an "effective amount" of the compositions described in the present disclosure refers to an amount required to elicit an immune response in the subject to which the composition was administered. The immune response is characterized by the presence of one or more Streptococcus pneumoniae antigen- specific antibodies in the host that significantly reduce the likelihood or severity of infection of Streptococcus pneumoniae during a subsequent challenge.

EXAMPLES

The following examples are provided to illustrate the invention and are merely for illustrative purposes only and should not be construed to limit the scope of the invention.

Example 1: Preparation of Pneumococcal Capsular Polysaccharide-CRMi97 Conjugates

Polysaccharide CRM197 conjugates for pneumococcal serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F were prepared as per the procedure described in PCT publication No. WO 2016/207905. Polysaccharide CRM197 conjugates for pneumococcal serotypes 6A, 6C, 15A, 15C, 23 A, 23B, 24F and 35B were prepared as per the procedure mentioned below:

a) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 6 A with CRM 197 protein using CDAP chemistry.

lOOOmg (68.5mL of 14.6mg/mL concentration) mechanically size reduced polysaccharide serotype 6A and 5.0mL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0:0.5(PS: CDAP) and stirred for 1 min. The pH of the polysaccharide solution was adjusted to 9.0 with 8.0mL of 0.2M triethylamine and stirred for 1 min at room temperature (RT). lOOOmg of CRM 197 (66.7mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs:CRM).

The pH of the reaction was adjusted to 9.0 with l.OmL of 0.2M triethylamine and the reaction was continued under stirring for 3 - 5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 2a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

The reaction mixture was diafiltered and concentrated using 100 kDa MWCO TFF membrane. Concentrate was purified by size-exclusion chromatography. The fractions were analyzed by SEC-MALLS, anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2 pm filters.

b) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 6C with CRM 197 protein using CDAP chemistry.

lOOOmg (100. OmL of lO.Omg/mL concentration) of mechanically size reduced polysaccharide serotype 6C and lO.OmL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.0 (PS:CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 15.5 mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of CRM197 (66.6mL of 15.0 mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs:CRM).

The pH of the reaction was adjusted to 9.0 with 2.0 mL of 0.2 M triethylamine and the reaction was continued under stirring for 3 - 5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 3a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

c) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 15 A with CRMm protein using CDAP chemistry.

1000 mg (66.7mL of 15.0mg/mL concentration) mechanically size reduced polysaccharide serotype 15A and lO.OmL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.0 (PS: CDAP) and stirred for 1 min. The pH of the polysaccharide solution was adjusted to 9.0 with 18.0mL of 0.2M triethylamine and stirred for 1 min at room temperature (RT). lOOOmg of CRM 197 (53.3mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0:0.8 (PnPs: CRM).

The pH of the reaction was adjusted to 9.0 with l.OmL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Ligure 4a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

The reaction mixture was diafiltered and concentrated using 100 kDa MWCO TEL membrane. Concentrate was purified by size-exclusion chromatography. The fractions were analyzed by SEC-MALLS, anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2pm filters.

d) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 15C with CRMm protein using CDAP chemistry.

lOOOmg (100. OmL of lO.Omg/mL concentration) of mechanically size reduced polysaccharide serotype 15C and 15. OmL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.5 (PS:CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 24.0mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of CRM197 (66.6mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs:CRM).

The pH of the reaction was adjusted to 9.0 with 2.8mL of 0.2M triethylamine and the reaction was continued under stirring for 3 - 5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Ligure 5a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

e) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 23 A with CRMm protein using CDAP chemistry.

lOOOmg (83.3mL of 15.0mg/mL concentration) mechanically size reduced polysaccharide serotype 23 A and lO.OmL of CDAP (100 mg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.0 (PS:CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 14.9mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). 800mg of CRM197 (53.3mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0:0.8 (PnPs:CRM).

The pH of the reaction was adjusted to 9.0 with 1.7mL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Ligure 6a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

The reaction mixture was diafiltered and concentrated using 100 kDa MWCO TEL membrane. Concentrate was purified by size-exclusion chromatography. The fractions were analyzed by SEC-MALLS, anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2 pm filters.

f) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 23 B with CRMm protein using CDAP chemistry.

1000 mg (100. OmL of lO.Omg/mL concentration) of mechanically size reduced polysaccharide serotype 23B and 2. OmL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0:0.2 (PS: CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 3.5mL of 0.2M triethylamine and stirred for 1 minute at room temperature (RT). lOOOmg of CRM197 (66.7mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs: CRM).

The pH of the reaction was adjusted to 9.0 with 2.2mL of 0.2M triethylamine and the reaction was continued under stirring for 3 - 5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Ligure 7a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

g) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 24F with CRMm protein using CDAP chemistry.

1000 mg (100.0 mL of lO.Omg/mL concentration) of mechanically size reduced polysaccharide serotype 24L and 5.0mL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0:0.5 (PS: CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 10.6mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). 800mg of CRM197 (53.3mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0:0.8 (PnPs: CRM).

The pH of the reaction was adjusted to 9.0 with l.OmL of 0.2M triethylamine and the reaction was continued under stirring for 3 - 5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Ligure 8a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

h) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 35 B with CRMm protein using CDAP chemistry.

1000 mg (100. OmL of lO.Omg/mL concentration) mechanically size reduced polysaccharide serotype 35B and 5. OmL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0:0.5 (PS:CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 5.0mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of CRM197 (66.7mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs:CRM).

The pH of the reaction was adjusted to 9.0 with 1.6mL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Ligure 9a) of reactions were monitored using SEC- HPLC at each hour of the reaction.

The reaction mixture was diafiltered and concentrated using 100 kDa MWCO TEL membrane. Concentrate was purified by size-exclusion chromatography. The fractions were analysed by SEC-MALLS, anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2pm filters.

Example 2: Formulation of Pneumococcal Capsular Polysaccharide-protein conjugate vaccine

A 20 valent conjugated vaccine was formulated as 0.5 mL dose containing 3 pg of Serotype 1, 2.2 pg of each pneumococcal polysaccharide from serotypes 3, 4, 5, 6A, 7L, 9V, 14, 18C, 19 A, 19L, 23L, 6C, 15A, 15C, 23A, 23B, 24L and 35B and 4.4 pg of 6B conjugated to ~ 45-50 pg of CRM 197 Protein. All the conjugates were adsorbed on to aluminum phosphate gel equivalent to 0.5 mg Al³⁺ per dose of 0.5 mL. The 0.9% W/V saline was used as diluent and vehicle for the formulation and the final formulation pH was adjusted to pH 6 using 1 N hydrochloric acid. Lor effective adsorption post adjusting the pH, the formulation was mixed for 2 hours under constant stirring. After 2 hours of blending, the formulated blend was aseptically filled at 0.58 mL fill volume per vial into the 3 mL sterile nonsiliconized vials, closed with sterile 13 mm rubber stoppers and sealed with 13 mm sterile pink colored flip off aluminum seals, followed by optical inspection and labelling of filled vials. Lrom the lot, some vials were randomly picked up and analyzed for the appearance, pH, Osmolality, total poly and protein content (pg/SHD), %Adsorption, aluminum content (mg/SHD) as given in Table 1 below. The product appearance is Whitish suspension in which the mineral carriers tends to settle down slowly.

Table 1: Formulation Testing Data

Example 3: Conjugation of individual pneumococcal polysaccharide to carrier protein to form Polysaccharide-PsaA Conjugates

PsaA Preparation, Activation and Conjugation of PsaA with Pneumococcal Polysaccharide Serotype 3, 6A and 6B was prepared as per the disclosure of PCT Publication WO 2018/064444 Al.

A) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 6C with PsaA protein using CDAP chemistry. lOOOmg (100. OmL of lO.Omg/mL concentration) of mechanically size reduced polysaccharide serotype 6C and lO.OmL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.0 (PS:CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 15.5mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of PsaA (66.6mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs: PsaA).

The pH of the reaction was adjusted to 9.0 with 2. OmL of 0.2M triethylamine and the reaction was continued under stirring for 3 - 5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 3b) of reactions were monitored using SEC- HPLC at each hour of the reaction.

The reaction mixture was diafiltered and concentrated using 100 kDa MWCO TFF membrane. Concentrate was purified by size-exclusion chromatography. The fractions were analyzed by SEC-MALLS, anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2pm filters.

B) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 15A with PsaA protein using CDAP chemistry.

lOOOmg (71.4mL of 14.0mg/mL concentration) mechanically size reduced polysaccharide serotype 15A and lO.OmL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.0 (PS:CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 20.5mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of PsaA (66.6mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs:PsaA).

The pH of the reaction was adjusted to 9.0 with 0.9mL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 4b) of reactions were monitored using SEC- HPLC at each hour of the reaction.

C) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 15C with PsaA protein using CDAP chemistry. 1000 mg (100.0 mL of lO.Omg/mL concentration) of mechanically size reduced polysaccharide serotype 15C and 15.0mL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.5 (PS:CDAP) and stirred for 1 Min. The pH of the polysaccharide solution was adjusted to 9.0 with 24.0mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of PsaA (66.6mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs: PsaA).

The pH of the reaction was adjusted to 9.0 with 2.8 mL of 0.2 M triethylamine and the reaction was continued under stirring for 3 - 5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 5b) of reactions were monitored using SEC- HPLC at each hour of the reaction.

D) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 23A with PsaA protein using CDAP chemistry.

lOOOmg (83.3mL of 12.0mg/mL concentration) mechanically size reduced polysaccharide serotype 23A and lO.OmL of CDAP (100 mg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0: 1.0 (PS:CDAP) and stirred for 1 min. The pH of the polysaccharide solution was adjusted to 9.0 with 20.3mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). 600mg of PsaA (40.0mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0:0.6 (PnPs:PsaA).

The pH of the reaction was adjusted to 9.0 with l.lmL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 6b) of reactions were monitored using SEC- HPLC at each hour of the reaction.

E) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 23 B with PsaA protein using CDAP chemistry. lOOOmg (100. OmL of lO.Omg/mL concentration) mechanically size reduced polysaccharide serotype 23B and 2.0 mL of CDAP (100 mg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0:0.2 (PS:CDAP) and stirred for 1 min. The pH of the polysaccharide solution was adjusted to 9.0 with 3. OmL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of PsaA (66.6mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs:PsaA).

The pH of the reaction was adjusted to 9.0 with 2.4 mL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 7b) of reactions were monitored using SEC- HPLC at each hour of the reaction.

F) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 24F with PsaA protein using CDAP chemistry.

lOOOmg (125. OmL of 8.0mg/mL concentration) mechanically size reduced polysaccharide serotype 24F and 3. OmL of CDAP (100 mg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0:0.3 (PS:CDAP) and stirred for 1 min. The pH of the polysaccharide solution was adjusted to 9.0 with lO.OmL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). 600mg of PsaA (40.0mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0:0.6 (PnPs:PsaA).

The pH of the reaction was adjusted to 9.0 with 3.5mL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 8b) of reactions were monitored using SEC- HPLC at each hour of the reaction.

G) Activation and Conjugation of Pneumococcal Polysaccharide Serotype 35B with PsaA protein using CDAP chemistry. lOOOmg (142.8mL of 7.0mg/mL concentration) mechanically size reduced polysaccharide serotype 35B and 6.0mL of CDAP (lOOmg/mL in Acetonitrile (w/v)) was mixed in a glass bottle in the ratio of 1.0:0.6 (PS:CDAP) and stirred for 1 min. The pH of the polysaccharide solution was adjusted to 9.0 with 7.0mL of 0.2M triethylamine and stirred for 1 Min at room temperature (RT). lOOOmg of PsaA (66.6mL of 15.0mg/mL concentration) was added slowly to the activated polysaccharide in a ratio of 1.0: 1.0 (PnPs:PsaA).

The pH of the reaction was adjusted to 9.0 with 2.2mL of 0.2M triethylamine and the reaction was continued under stirring for 3-5 hours at room temperature followed by quenching of the reaction by adding excess concentration of glycine (100 mM). The conjugation kinetics (Figure 9b) of reactions were monitored using SEC- HPLC at each hour of the reaction.

Example 4: Formulation of Pneumococcal Capsular Polysaccharide-protein conjugate vaccine

A 20 valent conjugated vaccine was formulated as 0.5 mL dose containing 3 pg of serotype 1, 2.2 pg of each pneumococcal polysaccharide from serotypes 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 15C, and 4.4 pg of 6B conjugated to ~ 30-45 pg of CRM197 protein and conjugates of pneumococcal polysaccharide from serotypes 3, 6A„ 15A, 23 A, 23B, 24F and 35B with PsaA as disclosed in above examples. All the conjugates were adsorbed on to aluminum phosphate gel equivalent to 0.5 mg Al³⁺ per dose of 0.5 mL. The 0.9% W/V saline was used as diluent and vehicle for the formulation and the final formulation pH was adjusted to pH 6 using IN hydrochloric acid. For effective adsorption post adjusting the pH, the formulation was mixed for 2 hours under constant stirring. After 2 hours of blending, the formulated blend was aseptically filled at 0.58 mL fill volume per vial into the 3 mL sterile nonsiliconized vials, closed with sterile 13 mm rubber stoppers and sealed with 13 mm sterile pink colored flip off aluminum seals, followed by optical inspection and labelling of filled vials. From the lot, some vials were randomly picked up and analyzed for the appearance, pH, Osmolality, total poly and protein content (pg/SHD), % Adsorption, aluminum content (mg/SHD) as given in Table 2 below. The product appearance is Whitish suspension in which the mineral carriers tends to settle down slowly. Table 2: Formulation Testing Data

Example 5: Formulation of Pneumococcal Capsular Polysaccharide-protein conjugate vaccine

A 20 valent conjugated vaccine was formulated as 0.5 mL dose containing 3 mg of serotype 1, 2.2 pg of each pneumococcal polysaccharide from serotypes, 4, 5, 7F, 9V, 14, 18C, 19 A, 19F, 23F, 6C, 15A, 15C, 23A, 23B, 24F and 35B conjugated to ~ 35-45 pg of CRM197 protein and conjugates of pneumococcal polysaccharide from serotypes 3, 6A and 6B with PsaA as disclosed in above examples. All the conjugates were adsorbed on to aluminum phosphate gel equivalent to 0.5 mg Al³⁺ per dose of 0.5 mL. The 0.9% W/V saline was used as diluent and vehicle for the formulation and the final formulation pH was adjusted to pH 6 using IN hydrochloric acid. For effective adsorption post adjusting the pH, the formulation was mixed for 2 hours under constant stirring. After 2 hours of blending, the formulated blend was aseptically filled at 0.58 mL fill volume per vial into the 3 mL sterile nonsiliconized vials, closed with sterile 13 mm rubber stoppers and sealed with 13 mm sterile pink colored flip off aluminum seals, followed by optical inspection and labelling of filled vials. From the lot, some vials were randomly picked up and analyzed for the appearance, pH, Osmolality, total poly and protein content (pg/SHD), % Adsorption, aluminum content (mg/SHD) as given in Table 2 below. The product appearance is Whitish suspension in which the mineral carriers tends to settle down slowly.

Table 3: Formulation Testing Data

Example 6: Immunogenicitv assessment of Pneumococcal conjugate formulations (PCV20V) in New Zealand White Rabbits

Immune response study was performed in New Zealand White rabbits. A 20 valent PCV formulation as described in Example 2 containing polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 6C, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23A, 23B, 23F, 24F and 35B conjugated to CRM197 was tested in rabbits. a) Immunization of rabbits:

Rabbits were immunized (seven rabbits) with single human dose on days 1, 15 and 29. They were bled on days 0, 14, and 28 while the final bleed was collected on day 36. The sera samples received at different time points under frozen condition were aseptically aliquoted in sterile vials, labeled appropriately and stored frozen at -80°C.

b) Determination of serotype-specific IgG titers:

Serotype specific antibody response was measured by Indirect ELISA. Briefly, specific pneumococcal polysaccharides received from ATCC, USA and SSI, Denmark were coated on Maxisorp ELISA plates and the unsaturated sites were blocked. The sera samples were pre adsorbed with CWPS Multi, added to the plate and diluted serially. Recombinant protein A/G peroxidase was added as the detection reagent. Finally, the color was developed using the HRP substrate, TMB. The reaction was stopped with 1.25M H2SO4 solution and absorbance was read at 450 nm in ELISA plate reader.

IgG geometric mean titers were calculated for pre-immune and day 36 sera of all the groups for all the different serotypes. Geometric mean fold rise was also calculated in day 36 sera of all the serotypes from different groups. The results obtained after immunizing with the formulation disclosed in Example 2 is represented in Figure 10. The IgG titer value of pre vaccinated animal was used to calculate Geometric Mean Fold Rise (GMFR) in serum IgG titer. The GMFR titer values were plotted in a graph (Figure 11).

The serotype- specific IgG titers were significantly high in all the groups as compared to pre-immune with geometric mean fold rise more than 4 for all the serotypes tested across different groups. The experiment indicates that the composition containing different formulation is immunogenic in rabbits.

From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention.

Claims

The claims:

1. A vaccine composition comprising Streptococcus pneumoniae capsular polysaccharide protein conjugates, wherein each conjugate comprises a polysaccharide from a Streptococcus pneumoniae serotype conjugated to a carrier protein, and wherein the vaccine composition comprises capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C or 6D, 15A, 15C, 23A, 23B, 24F and 35B.

2. The vaccine composition as claimed in claim 1, wherein the carrier protein is selected from a group comprising CRM197, PsaA, combination of CRM197 and PsaA, combination of CRM197 and Tetanus toxoid, combination of PsaA and Tetanus toxoid, and combination of CRM197, PsaA and Tetanus toxoid.

3. A 20-valent pneumococcal conjugate vaccine composition comprising capsular polysaccharide from serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the serotypes are selected from a group comprising 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19 A, 19F, 23F,6C or 6D, 15A, 15C, 23A, 23B, 24F and 35B,

and wherein, the carrier protein is selected from a group comprising CRM197, PsaA, combination of CRM197 and PsaA, combination of CRM197 and Tetanus toxoid, combination of PsaA and Tetanus toxoid, and combination of CRM197, PsaA and Tetanus toxoid.

4. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B, and wherein the carrier protein is CRM197.

5. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B, and wherein the carrier protein is PsaA.

6. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B, and wherein the carrier protein is selected from a group comprising CRM197, PsaA and combinations thereof.

7. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19 A, 19F, 23F, 23A, 23B, 24F and 35B,

and wherein at least 13 polysaccharides are conjugated to first carrier protein, at least 3 polysaccharides are conjugated to second carrier protein and at least one polysaccharide is conjugated to a third carrier protein, and wherein the first, second and third carrier proteins are selected from a group comprising CRM197, PsaA and Tetanus toxoid.

8. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B, and wherein at least 13 polysaccharides are conjugated to first carrier protein and at least 3 polysaccharides are conjugated to second carrier protein, and wherein the first and second carrier proteins are CRM197 and Tetanus toxoid.

9. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B, and wherein at least 13 polysaccharides are conjugated to first carrier protein, at least 3 polysaccharides are conjugated to second carrier protein, wherein the first and second carrier proteins are CRM₁₉₇ and PsaA.

10. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent vaccine composition comprising capsular polysaccharide from Streptococcus pneumonia serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C or 6D, 15C and 6B conjugated to CRM₁₉₇ and capsular polysaccharide from Streptococcus pneumonia serotypes 3, 6A,15A, 23A, 23B, 24F and 35B conjugated to PsaA.

11. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent vaccine composition comprising capsular polysaccharide from Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C or 6D, 15A, 15C, 23A, 23B, 24F and 35B conjugated to CRM197 and capsular polysaccharide from Streptococcus pneumonia serotypes 3, 6 A and 6B conjugated to PsaA.

12. The vaccine composition as claimed in claim 3, wherein the composition is a 20 valent pneumococcal conjugate vaccine composition comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C or 6D, 7F, 9V, 14, 15A, 15C, 18C, 19A, 19F, 23F, 23A, 23B, 24F and 35B, and wherein the carrier protein is CRM197, PsaA and Tetanus toxoid, and wherein each polysaccharide conjugate has a molecular weight ranging between about 1,000 kDa to about 15,000 kDa and the ratio of protein to polysaccharide (protein/PS) is in the range from 0.5 to 1.5.

13. The vaccine composition as claimed in any of the preceding claims, wherein the composition further comprises one or more pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a buffer, a preservative, a stabilizer, an adjuvant, and/or a lyophilization excipient.

14. A method of inducing an immune response to Streptococcus pneumoniae comprising administering an immunologically effective amount of the vaccine composition as claimed in any one of claims 1-13.