RU2801304C2 - Pneumococcal polysaccharides and their use in polysaccharide-protein-carrier immunogenic conjugates - Google Patents

Abstract

FIELD: pharmaceuticals; immunology.

SUBSTANCE: group of inventions relates to capsular polysaccharides from Streptococcus pneumoniae serotypes. A polysaccharide-carrier protein conjugate is disclosed, where the polysaccharide contains a repeating unit of the specified structure, where each x independently denotes a molar ratio of 0–1.0; wherein the carrier protein is CRM197 and wherein the polysaccharide-carrier protein conjugate has a polysaccharide to carrier protein weight ratio of 0.5–2.0. Besides, the invention relates to polysaccharide-protein conjugates in which capsular polysaccharides from one or more of these serotypes are conjugated to a carrier protein such as CRM197. Moreover, an alternative polysaccharide-carrier protein conjugate is disclosed, wherein the carrier protein is CRM197 and wherein the polysaccharide-carrier protein conjugate has a molecular weight of 1,000–10,000 kDa.

EFFECT: group of inventions provides effective immunogenic pneumococcal compositions.

26 cl, 9 dwg, 1 tbl, 7 ex

Description

FIELD OF THE INVENTION

The present invention relates to purified capsular polysaccharides from Streptococcus pneumoniae serotype 31 and polysaccharide-protein conjugates having polysaccharides from this serotype. Polysaccharide-protein conjugates from this serotype can be included in multivalent pneumococcal conjugate vaccines.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae , one example of an encapsulated bacterium, is a significant cause of serious disease worldwide. In 1997, the Centers for Disease Control and Prevention (CDC) estimated that there were 3,000 cases of pneumococcal meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000 cases of pneumococcal otitis media, and 500,000 cases of pneumococcal pneumonia annually in the United States. See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997, 46(RR-8):1-13. In addition, the complications of these diseases can be significant, with some studies reporting up to 8% mortality and 25% neurological complications in pneumococcal meningitis. See Arditi et al ., 1998, Pediatrics 102:1087-97.

Multivalent pneumococcal polysaccharide vaccines, licensed for many years, have been shown to be invaluable in preventing pneumococcal disease in adults, particularly the elderly and those at high risk. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T-cell independent immunogens that elicit a weak or no response in infants. Chemical conjugation of a bacterial polysaccharide immunogen to a carrier protein translates the immune response into a T-cell dependent response in infants. Diphtheria toxoid (DTx, a chemically detoxified variant of DT) and CRM197 have been described as carrier proteins for bacterial polysaccharide immunogens due to the presence of T cell stimulating epitopes in their amino acid sequences.

The pneumococcal conjugate vaccine, ^Prevnar® , containing the 7 most commonly isolated serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) causing invasive pneumococcal disease in infants and infants at the time, licensed for the first time in the United States in February 2000. Following the universal use of ^Prevnar® in the United States, there has been a significant reduction in the incidence of invasive pneumococcal disease in children due to the serotypes present in ^Prevnar® . See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005, 54(36):893-7. However, there are limitations of serotype overlap using Prevnar ^® in certain areas of the world, and some evidence for the emergence of certain serotypes in the United States (eg 19A and others). See O'Brien et al ., 2004, Am J Epidemiol 159:634-44; Whitney et al ., 2003, N Engl J Med 348:1737-46; Kyaw et al ., 2006, N Engl J Med 354:1455-63; Hicks et al ., 2007, J Infect Dis 196:1346-54; Traore et al ., 2009, Clin Infect Dis 48:S181-S189.

Prevnar ^13® is a 13-valent pneumococcal polysaccharide protein conjugate vaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. See, for example, U.S. Patent Application Publication No. US 2006/0228380 A1, Prymula et al ., 2006, Lancet 367:740-48 and Kieninger et al ., Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given as a 4 -Dose Series in Healthy Infants and Toddlers, presented at the ^48th Annual ICAAC/ISDA ^46th Annual Meeting, Washington DC, October 25-28, 2008. See also Dagan et al ., 1998, Infect Immun. 66:2093-2098 and Fattom, 1999, Vaccine 17:126.

S. pneumoniae is categorized from over ninety serotypes based on the structure of the capsular polysaccharide. For a list of known structures of pneumococcal capsular polysaccharides, see Geno, 2015, Clinical Microbiology Reviews 28:871–899. It has now been determined that the previously published structure for serotype 31 (Kamerling, 2000, Pneumococcal polysaccharides: a chemical view. In Tomasz A (ed), Streptococcus pneumoniae molecular biology & mechanisms disease. Mary Ann Liebert, Inc., Larchmont, NY. pp. 81–114) is incorrect.

Modern multivalent pneumococcal conjugate vaccines have been effective in reducing the incidence of pneumococcal disease associated with those serotypes present in the vaccines. However, the prevalence of pneumococci expressing serotypes not present in the vaccine has increased. Accordingly, there is a need to identify and characterize emerging pneumococcal serotypes for inclusion in future vaccines.

SUMMARY OF THE INVENTION

The present invention relates to purified capsular polysaccharides from Streptococcus pneumoniae serotype 31 and polysaccharide-protein conjugates having this serotype. The present invention is based, in part, on the structural identification of capsular polysaccharides from this serotype.

Accordingly, in one embodiment, the present invention relates to a polysaccharide with the following repeat unit:

where each x is independently a molar ratio from 0.0 to 1.0, and the OAc group on Gal f may be present in either the 5 or 6 position, or both.

A polysaccharide from Streptococcus pneumoniae serotype 31 can be represented by the following formula

, where n is the number of repeating links.

In specific embodiments, the polysaccharide has between 10 and 5000 repeat units. In specific aspects, the polysaccharide has between 25 and 3000, 100 and 2500, 100 and 1000, and 100 and 500 repeat units.

In specific embodiments, the polysaccharide has a molecular weight of 100 kDa to 4000 kDa, or 100 kDa to 3000 kDa. In specific aspects, the polysaccharide has a molecular weight of 100 kDa to 2000 kDa, 200 kDa to 5000 kDa, or 100 kDa to 250 kDa.

In specific embodiments, the S. pneumoniae serotype 31 polysaccharide has a molar ratio of O-acetyl groups to serotype 31 repeat unit from 0.0 to 3.0, e.g., any of 0.1, 0.2, 0.3, 0, 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2, 9, up to 3.0. Due to the availability of three positions where OAc can bind, molar ratios up to 3.0 are available.

In addition, the present invention relates to activated polysaccharides obtained in any of the above embodiments, where the polysaccharide is activated using a chemical reagent to obtain reactive groups for conjugation with a linker or carrier protein.

In addition, the present invention relates to polysaccharide-protein conjugates in which the polysaccharides or activated polysaccharides as described above are conjugated to a carrier protein. In specific aspects, the carrier protein is selected from CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, E. coli LT, ST E. coli and exotoxin A from Pseudomonas aeruginosa . In one specific aspect, the carrier protein is CRM197.

In specific aspects, polysaccharide-protein conjugates are prepared using reductive amination chemical reactions under aqueous conditions or in an aprotic solvent such as dimethyl sulfoxide (DMSO). In a specific aspect, polysaccharide-protein conjugates are prepared using reductive amination chemistries in DMSO.

In one embodiment, the present invention provides a multivalent immunogenic composition comprising unconjugated polysaccharides or polysaccharide-protein conjugates from Streptococcus pneumoniae serotype 31 and unconjugated polysaccharides or polysaccharide-protein conjugates from one or more of Streptococcus pneumoniae serotypes 1, 2, 3, 4 , 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20 , 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 28A, 33F, 34, 35A, 35B, 35F and 38. In one sub-embodiment, the multivalent immunogenic composition comprises unconjugated polysaccharides or polysaccharide-protein- carrier, but not both. In one sub-embodiment, the multivalent immunogenic composition comprises a mixture of unconjugated polysaccharides or polysaccharide-carrier protein conjugates. In specific sub-embodiments, the multivalent immunogenic composition of the invention has up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 serotypes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows graphical representations of the structure of repeating units of the S. pneumoniae serotype 31 polysaccharide.

The figure 2 shows the spectrum of one-dimensional ¹ H NMR at 600 MHz for capsular polysaccharide from S. pneumoniae serotype 31 in D ₂ O at 50°C. Signals from internal standards (DMSO and DSS-d6), residual water (HOD) and other residual components of the purification process are noted; ethanol (EtOH), isopropanol (IPA) and acetate. Minor signals marked with * are due to S. pneumoniae cell wall residues such as C-polysaccharide and/or peptidoglycans.

Figure 3 shows the one-dimensional (1D) ¹ H NMR region of identity for use in identifying serotypes of S. pneumoniae serotype 31. Signal positions of each anomeric proton of repeat units from a monosaccharide residue are marked.

Figure 4 shows a partial two-dimensional (2D) NMR spectrum for multiple ¹ H- ¹³ C linkage correlation of a de-O-acetylated S. pneumoniae serotype 31 polysaccharide forming covalent bonds between sugar moieties in a repeat pattern. Correlating glycosidic bonds are marked in the figure.

Figure 5 shows a partial 2D ¹ H - ¹³ C NMR spectrum for multiple linkage correlation for a purified S. pneumoniae serotype 31 polysaccharide forming O-acetate linkages.

Figure 6: ELISA titers of IgG antibodies (after dosing 2) for rabbits immunized with monovalent S. pneumoniae serotypes conjugated to CRM197 and formulated with aluminum phosphate (APA) adjuvant. Error bars represent the geometric mean + 95% confidence interval.

Figure 7: OPA serotype-specific titers (after dosing 2) for rabbits immunized with monovalent S. pneumoniae serotypes conjugated to CRM197 and formulated with aluminum phosphate (APA) adjuvant. Error bars represent the geometric mean + 95% confidence interval.

Figure 8 shows geometric mean titers of serotype-specific ( S. pneumoniae serotypes 16F, 23A, 23B, 24F, 31) antibodies, pre-immunization, PD1 and PD2, for rabbits immunized with a multivalent pneumococcal conjugate vaccine (2 μg/PnPs) . Error bars represent 2 standard errors of the geometric mean titer of each serotype (X-axis).

Figure 9 shows dilution titers of serotype-specific ( S. pneumoniae serotypes 16F, 23A, 23B, 24F, 31) OPA, pre-immunization, PD1 and PD2, for rabbits immunized with a multivalent pneumococcal conjugate vaccine (2 μg/PnPs). Symbols denote individual titers and error bars represent 95% confidence intervals (CI) of geometric mean titers (GMT).* p<0.05, ** p<0.01, *** p<0.001, ns=not significant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the identification of novel pneumococcal polysaccharide structure(s) by means of NMR technology. The structure presented herein is believed to be the first identification or the first correct identification for S. pneumoniae serotype 31.

The S. pneumoniae serotype 31 polysaccharide was produced in its respective strain and purified. The produced (and purified) polysaccharides were used to prepare individual Ps-CRM197 conjugates. S. pneumoniae serotype 31 has a unique polysaccharide structure resulting in a unique conjugate preparation process. The resulting conjugate(s) was shown to be immunogenic in animal studies.

For the purposes of the invention, the term "polysaccharide" (Ps) is understood to include any antigenic saccharide element (or antigenic unit) generally accepted in the field of immunology and bacterial vaccines, including, but not limited to, "saccharide", "oligosaccharide", "polysaccharide", "liposaccharide", "lipooligosaccharide (LOS)", "lipopolysaccharide (LPS)", "glycosylate", "glycoconjugate", "derivatized or activated polysaccharide or oligosaccharide", and the like. Unless otherwise indicated, the nomenclature of polysaccharides within the scope of the invention follows the recommendations of the Joint Commission on Biochemical Nomenclature IUB-IUPAC (JCBM) 1980. See JCBN, 1982, J. Biol. Chem. 257:3352-3354.

As used herein, "immunogenic composition" refers to a composition containing an antigen, such as a bacterial capsular polysaccharide or a polysaccharide-protein conjugate, having the ability to elicit an immune response, either humoral or cell-mediated, or both, in a host, such as a mammal. The immunogenic composition may serve to sensitize the host by presenting the antigen in association with MHC molecules on the cell surface. In addition, antigen-specific T cells or antibodies can be generated to provide future protection to the immunized host. Immunogenic compositions may thus protect the host from bacterial infection, reduce the severity, or may protect the host from death due to bacterial infection. Immunogenic compositions can also be used to generate polyclonal or monoclonal antibodies that can be used to confer passive immunity in a subject. Immunogenic compositions can also be used to generate antibodies that are functional as measured by bacterial kill in either an animal efficacy model or opsonophagocytic kill assay.

In the context of the invention, the term "isolated", when applied to a polysaccharide, refers to the isolation of a serotype-specific S. pneumoniae capsular polysaccharide from a purified polysaccharide using purification methods known in the art, including the use of centrifugation, depth filtration, precipitation, ultrafiltration, treatment with using activated carbon, diafiltration and/or column chromatography. Generally, isolated polysaccharide refers to the partial removal of proteins, nucleic acids, and a non-specific endogenous polysaccharide (C-polysaccharide). The isolated polysaccharide contains less than 10%, 8%, 6%, 4%, or 2% protein contaminants and/or nucleic acids. The isolated polysaccharide contains less than 20% C-polysaccharide relative to type-specific polysaccharides.

In the context of the invention, the term "purified" in relation to a bacterial capsular polysaccharide refers to the purification of the polysaccharide from a cell lysate by methods such as centrifugation, precipitation and ultrafiltration. Generally, purified polysaccharide refers to the removal of cellular debris and DNA.

Within the scope of the invention, the term "Mw" refers to the weight average molecular weight and is generally expressed in Da or kDa. Mw takes into account that the larger molecule contains more of the total mass of the polymer sample than the smaller molecules. Mw can be determined by methods such as static light scattering, small angle neutron scattering, x-ray scattering, and sedimentation rate.

Within the scope of the invention, the term "Mn" refers to the number average molecular weight and is typically expressed in Da or kDa. Mn is calculated by obtaining the total mass of the sample divided by the number of molecules in the sample, and can be determined by methods such as gel permeation chromatography, viscometry by (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, determination of end groups or proton NMR. Mw/Mn reflects polydispersity.

Within the scope of the invention, the term "molar ratio" is a fraction, usually expressed as a decimal fraction to the first or second decimal place. For example, a molar ratio from 0 or 0.1 to 1.0, expressed to the first decimal place, may include any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.

Within the scope of the invention, the abbreviation "PnPs" refers to pneumococcal polysaccharide.

Within the scope of the invention, the term "comprises", when used in relation to an immunogenic composition of the invention, refers to the inclusion of any other components (to be limited to "consisting of" for a mixture of antigens), such as adjuvants and excipients. The term "consisting of" in relation to the multivalent mixture of polysaccharide-protein conjugates of the present invention refers to a mixture containing these particular polysaccharide conjugatesS. pneumoniae with protein, not other conjugates with polysaccharide proteinS. pneumoniae from another serotype.

Within the meaning of the invention, the phrase "activation site" on a sugar means that the site can be chemically modified to form a reactive group. The site of activation takes into account the preferred tendency of the activating agent to react with a particular site.

In the context of the invention, the phrase "activated polysaccharide" refers to a polysaccharide that has been chemically modified to form reactive groups in the polysaccharide chain. An activated polysaccharide does not necessarily mean that all available activation sites have been chemically modified.

In the context of the invention, the phrase "degree of activation" on a polysaccharide chain refers to the overall ratio between the number of activated chemical group and the number of repeating units on the polysaccharide chain.

Unless otherwise noted, all ranges provided herein include the lower and upper limits listed.

In the examples, the structure of the S. pneumoniae serotype 31 polysaccharide is identified, which differs from the published structure. See Kamerling, 2000, Pneumococcal polysaccharides: a chemical view, p. 81-114. In Tomasz (ed), Streptococcus pneumoniae molecular biology & mechanisms of disease. Mary Ann Liebert, Inc., Larchmont, NY. The composition of the monosaccharide is consistent with the view that both structures have 2 Rha, 2 Gal and 1 GlcA. However, the Gal residues in the published structure are both furanose rings, while in the structure shown in the examples, the Gal residues are 1 furanose and 1 pyranose. The structure in the examples also has three acetate groups not present in the previously described structure. There is a possibility that this represents an additional subtype present among serogroup 31 due to two different chemical structures.

The polysaccharide structure of S. pneumoniae serotype 31 shows the presence of two O-acetyl groups on the β-Gal f saccharide and one O-acetyl group on the β-Rha p saccharide, at approximately 90% in each of the three regions. β-Gal f has O-acetyl groups at position 5 or 6 on the saccharide. In specific embodiments, the serotype 31 polysaccharide has, either in the β-Rha p saccharide or in any of the β-Gal f positions, 0 (i.e., fully de-O-acetylated) or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mM acetate per mmol serotype 31 polysaccharide.

The identification of the structure of these serotype(s) may allow their inclusion in pneumococcal vaccines, either unconjugated or in the form of a polysaccharide-protein conjugate. Conjugate vaccines containing streptococcal and pneumococcal Ps are well known in the art. See, for example, US Patent No. 6248570; 5866135; and 5773007.

Capsular polysaccharides

Capsular polysaccharides from Steptococcus pneumoniae from the serotype(s) of the invention can be obtained by standard methods known to those skilled in the art. For example, polysaccharides can be isolated from bacteria, and size correction can be carried out to some extent by known methods (see, for example, European patents No. EP497524 and EP497525); and preferably by microfluidization carried out using a homogenizer or by chemical hydrolysis. In one embodiment, S. pneumoniae strains corresponding to each polysaccharide serotype are grown in soy-based media. The individual polysaccharides are then purified by standard steps including centrifugation, precipitation and ultrafiltration. See, for example, U.S. Patent Application Publication No. 2008/0286838 and US Patent No. 5,847,112. The size of the polysaccharides can be adjusted to reduce the viscosity and/or to improve the filterability of subsequent conjugate products. Chemical hydrolysis can be carried out using acetic acid. Mechanical sizing can be carried out using cutting through high pressure homogenization.

In some embodiments, the purified polysaccharides prior to conjugation have a molecular weight between 5 kDa and 4000 kDa. Molecular weight can be calculated by size exclusion chromatography (SEC), in combination with a multi-angle light scattering (MALS) detector and a refractive index (RI) detector. In other such embodiments, the polysaccharide has an average molecular weight between 10 kDa and 4000 kDa; between 50 kDa and 4000 kDa; between 50 kDa and 3000 kDa; between 50 kDa and 2000 kDa; between 50 kDa and 1500 kDa; between 50 kDa and 1000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDa and 4000 kDa; between 100 kDa and 3000 kDa; 100 kDa and 2000 kDa; between 100 kDa and 1500 kDa; between 100 kDa and 1000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 100 and 400 kDa; between 200 kDa and 4000 kDa; between 200 kDa and 3000 kDa; between 200 kDa and 2000 kDa; between 200 kDa and 1500 kDa; between 200 kDa and 1000 kDa; or between 200 kDa and 500 kDa. In specific embodiments, the polysaccharide has a molecular weight of 100 kDa to 3000 kDa. In specific aspects, the polysaccharide has a molecular weight of 100 kDa to 4000 kDa, 100 kDa to 3000 kDa, 100 kDa to 2000 kDa, 200 kDa to 500 kDa, or 100 kDa to 250 kDa.

In specific embodiments, the polysaccharide has between 10 and 5000 repeat units. In specific aspects, the polysaccharide has between 25 and 3000, 100 and 2500, 100 and 1000, and 100 and 500 repeat units.

In specific embodiments, the S. pneumoniae serotype 31 polysaccharide has a molar ratio of O-acetyl groups to serotype 31 repeat units of 0.1-3.0, e.g., any of 0.1, 0.2, 0.3, 0.4 , 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1 .7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 , up to 3.0. Due to the availability of three positions where OAc can bind, molar ratios up to 3.0 are available.

carrier protein

Polysaccharides from one or more serotypes can be conjugated to a carrier protein ("Pr") to improve immunogenicity in children, the elderly and/or immunocompromised subjects. When more than one serotype is used in a multivalent composition, serotypes can be generated with the same carrier protein or different carrier proteins. Each capsular polysaccharide of the same serotype is usually conjugated to the same carrier protein.

In a specific embodiment of the present invention, CRM197 is used as a carrier protein. CRM197 is a non-toxic variant of diphtheria toxin (DT). The CRM197 carrier protein is a mutant form of DT that is rendered non-toxic by a single amino acid substitution at fragment A at residue 52. In one embodiment, the CRM197 carrier protein is isolated from Corynebacterium diphtheria strain C7 (β197) grown in a casamino acid-based medium. and yeast extract. In another embodiment, CRM197 is produced in a recombinant manner, in accordance with the methods described in US Patent No. 5,614,382. Typically, CRM197 is purified by a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, CRM197 is produced in Pseudomonas fluorescens using Pfenex Expression™ technology (Pfenex Inc., San Diego, CA).

Other useful carrier proteins include additional inactivated bacterial toxins such as DT, fragment B of diphtheria toxoid (DTFB), TT (tetanus toxoid) or fragment C of TT, pertussis toxoid, cholera toxoid (for example, as described in International Patent Application Publication No. WO 2004/083251), E. coli LT (thermolabile enterotoxin), E. coli ST (thermolabile enterotoxin), and Pseudomonas aeruginosa exotoxin A. Bacterial outer membrane proteins can also be used, such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; See International Patent Application Publication No. WO 02/091998), pneumococcal protein adhesin (PsaA ), peptidase C5a from group A or group B streptococcus, or Haemophilus influenzae protein D, pneumococcal pneumolysin (Kuo et al . GMBS (See International Patent Application Publication No. WO 04/081515) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and Pht fusion proteins, e.g. PhtDE fusion proteins, PhtBE fusion proteins (See Publication of international patent applications No. WO 01/98334 and WO 03/54007). Other proteins such as ovalbumin, limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivative (PPD), PorB (from N. meningitidis ), PD ( Haemophilus influenzae protein D; see e.g. European patent No. EP 0 594 610 B), or their immunological functional equivalents, synthetic peptides (See European patents No. EP0378881 and EP0427347), heat shock proteins (See International patent application publications No. WO 93/17712 and WO 94/ 03208), pertussis proteins (See International Patent Publication No. WO 98/58668 and European Patent No. EP0471177), cytokines, lymphokines, growth factors or hormones (See International Patent Publication No. WO 91/01146), artificial proteins containing multiple epitopes recognized by human CD4+ T cells from antigens derived from various pathogens (See Falugi et al ., 2001, Eur J Immunol 31:3816-3824) such as the N19 protein (See Baraldoi et al ., 2004, Infect Immun 72:4884-7), iron capture proteins (See Publication of International Patent Application No. WO 01/72337), toxin A or B from C. difficile (See International Patent Publication No. WO 00/61761) and flagellin (See Ben-Yedidia et al ., 1998, Immunol Lett 64:9), can also be use as carrier proteins.

Other DT mutants can also be used as carrier proteins such as CRM176, CRM228, CRM45 (Uchida et al. , 1973, J Biol Chem 218:3838-3844); CRM9, CRM45, CRM102, CRM103 and CRM107, and other mutations described in Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser, and/or Ala 158 to Gly, and other mutations described in US Patent No. 4709017 or US Patent No. 4950740; mutation of at least one or more residues of Lys 516, Lys 526, Phe 530 and/or Lys 534, and other mutations described in US Patent No. 5917017 or US Patent No. 6455673; or the fragment described in US Patent No. 5843711.

When using multivalent vaccines, the second carrier protein can be used for one or more antigens. The second carrier protein is preferably a protein which is non-toxic and non-reactogenic and can be obtained in sufficient quantity and with sufficient purity. The second carrier protein is also conjugated or linked to an antigen, such as a S. pneumoniae polysaccharide, to increase the immunogenicity of the antigen. Carrier proteins should be suitable for standard conjugation methods. In one embodiment, each capsular polysaccharide not conjugated to a first carrier protein is conjugated to the same second carrier protein (eg, each capsular polysaccharide molecule is conjugated to a single carrier protein). In another embodiment, the capsular polysaccharides not conjugated to the first carrier protein are conjugated to two or more carrier proteins (each capsular polysaccharide molecule is conjugated to one carrier protein). In such embodiments, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.

Conjugation

Prior to conjugation, the purified polysaccharides can be chemically activated to render the saccharides capable of reacting with a carrier protein to produce an activated polysaccharide. Within the scope of the invention, the term "activated polysaccharide" refers to a polysaccharide that has been chemically modified, as described below, to allow conjugation to a linker or carrier protein. Purified polysaccharides can optionally be combined with a linker. After activation or connection to a linker, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. Polysaccharide conjugates can be prepared by known coupling methods.

In specific embodiments, a polysaccharide can be attached to a linker to form a polysaccharide-linker intermediate wherein the free end of the linker is an ester group. The linker is thus a linker in which at least one end of the linker is an ester group. The other end is chosen such that it can react with the polysaccharide to form a polysaccharide-linker intermediate.

In specific embodiments, the polysaccharide can be attached to the linker using the primary amino group in the polysaccharide. In this case, the linker typically has an ester group at both ends. This allows addition by reaction of one of the ester groups with a primary amino group in the polysaccharide via nucleophilic acyl substitution. The reaction results in a polysaccharide-linker intermediate in which the polysaccharide is attached to the linker via an amide bond. The linker is thus a bifunctional linker providing a first ester group for reaction with the primary amino group on the polysaccharide and a second ester group for reaction with the primary amino group on the carrier molecule. A typical linker is an N-hydroxysuccinimide diester of adipic acid (SIDEA).

In specific embodiments, the attachment can also be done indirectly, ie, using an additional linker that is used to derivatize the polysaccharide prior to attachment to the linker. The polysaccharide is attached to an additional linker using a carbonyl group at the reducing end of the polysaccharide. This addition involves two steps: (a1) reaction of the carbonyl group with an additional linker; and (a2) reacting the free end of the additional linker with the linker. In these embodiments, the additional linker typically has an amino group at both ends, thus allowing step (a1) to be carried out by reacting one of the primary amino groups with a carbonyl group in the polysaccharide via reductive amination. A primary amino group is used which is reactive with respect to the carbonyl group in the polysaccharide. Hydrazide or hydroxylamine groups are suitable. The same primary amino group is typically present at both ends of the additional linker. The reaction results in a polysaccharide-additional linker intermediate in which the polysaccharide is linked to the additional linker via a C-N bond.

In specific embodiments, the polysaccharide can be attached to an additional linker using another group on the polysaccharide, in particular a carboxyl group. This addition involves two steps: (a1) reaction of the group with an additional linker; and (a2) reacting the free end of the additional linker with the linker. In this case, the additional linker typically has a primary amino group at both ends, thus allowing step (a1) to be carried out by reacting one of the primary amino groups with a carboxyl group in the polysaccharide via EDAC activation. A primary amino group is used that is reactive with the EDAC-activated carboxyl group in the polysaccharide. The hydrazide group is suitable. The same primary amino group is typically present at both ends of the additional linker. The reaction results in a polysaccharide-additional linker intermediate in which the polysaccharide is linked to the additional linker via an amide bond.

In one embodiment, chemical activation of polysaccharides and subsequent conjugation to a carrier protein via reductive amination can be performed by the methods described in US Patent Nos. 4,365,170, 4,673,574 and 4,902,506, U.S. Patent Application Publication Nos. 2006/0228380, 2007/184072, 2007/0231340 and 2007/0184071, and International Patent Application Publication No. WO2006/110381, WO2008/079653 and WO2008/143709). Chemical reactions may include activation of the pneumococcal polysaccharide by reaction with any primary hydroxyl oxidizing agent with an aldehyde such as TEMPO in the presence of an oxidizing agent (WO2104/097099), or reaction of the two vicinal hydroxyl groups with aldehydes such as periodate (including sodium periodate, potassium periodate or periodic acid). The reactions lead to random oxidation of primary hydroxyl groups or random oxidative cleavage of carbohydrate vicinal hydroxyl groups to form reactive aldehyde groups.

In this embodiment, attachment to the carrier protein occurs via reductive amination via direct amination of the lysyl groups of the protein. For example, conjugation is carried out by reacting a mixture of activated polysaccharide and carrier protein with a reducing agent such as sodium cyanoborohydride in the presence of nickel. The conjugation reaction can be carried out in an aqueous solution or in the presence of dimethyl sulfoxide (DMSO). See, for example, U.S. Patent Application Publication No. US2015/0231270 and US2011/0195086, and European patent no. EP 0471 177 B1. The unreacted aldehydes are then capped using the addition of a strong reducing agent such as sodium borohydride.

Reductive amination involves two steps, (1) oxidation of the polysaccharide to form reactive aldehydes, (2) reduction of the imine (Schiff base) formed between the activated polysaccharide and the carrier protein to form a stable amine bond of the conjugate. Prior to oxidation, the polysaccharide is optionally subjected to size reduction. You can use mechanical methods (for example, homogenization) or chemical hydrolysis. Chemical hydrolysis can be carried out using acetic acid. The oxidation step may include reaction with periodate. For the purposes of the present invention, the term "periodate" includes both periodate and periodic acid; the term also includes both metaperiodate (IO ₄ ^- ) and orthoperiodate (IO ₆ ^5- ), and includes various periodate salts (eg, sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate (NaIO ₄ ). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of a periodic acid.

In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxide radical compound, such as piperidine-N-oxy or pyrrolidine-N-oxy compounds, in the presence of an oxidizing agent to selectively oxidize primary hydroxyls (as described, for example, in Publication International Patent Application No. WO 2014/097099). In this reaction, the actual oxidizing agent in the catalytic cycle is the N-oxoammonium salt. In one aspect, said stable nitroxyl or nitroxide radical compound is piperidine-N-oxy or pyrrolidine-N-oxy compounds. In one aspect, said 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, said stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, said oxidizing agent is a molecule bearing an N-halogen group. In one aspect, said oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinan-2,4,6-trione , dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinan-2,4,6-trione, diiodioisocyanuric acid, 1,3,5-triiodo-1,3,5-triazinan-2,4 ,6-trion. Preferably, said oxidizing agent is N-chlorosuccinimide.

In specific aspects, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) as a co-oxidizing agent (as described in International Patent Application Publication No. WO2014/097099) . Thus, in one aspect, glycoconjugates from S. pneumoniae can be obtained by a method comprising the steps of: a) reacting the saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to obtain activated saccharide; and b) reacting the activated saccharide with a carrier protein containing one or more amino groups (this method is referred to hereinafter as "TEMPO/NCS reductive amination").

Optionally, the oxidation reaction is quenched by adding a quenching agent. The quenching agent may be selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites, or phosphorous acid (e.g., glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid).

The second step of the conjugation process in the case of reductive amination is the reduction of the imine bond (Schiff base) between the activated polysaccharide and the carrier protein to form a stable conjugate bond (so-called reductive amination), using a reducing agent. Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride) or sodium borohydride. In one embodiment, the reducing agent is sodium cyanoborohydride.

In specific embodiments of the methods of the invention, the reductive amination reaction is carried out in an aprotic solvent (or a mixture of aprotic solvents). In one embodiment, the reduction reaction is carried out in the solvent DMSO (dimethyl sulfoxide) or DMF (dimethylformamide). DMSO or DMF solvent can be used to dilute the activated polysaccharide and carrier protein if they have been lyophilized. In one embodiment, the aprotic solvent is DMSO.

At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugates, which can be capped or quenched using a suitable capping or quenching agent. In one embodiment, this capping or quenching agent is sodium borohydride (NaBH ₄ ). Suitable alternatives include sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of Bronsted or Lewis acids, aminoboranes such as pyridineborane, 2-picolineborane, 2,6-diboranemethanol, dimethylamineborane, t-BuMe'PrN-BH ₃ , benzylamine-BH ₃ or 5 -ethyl-2-methylpyridineborane (PEMB), or a borohydride exchange resin.

Glycoconjugates prepared using reductive amination in an aprotic solvent are generally used in multivalent pneumococcal conjugate vaccines. Thus, in specific embodiments of multivalent formulations where not all serotypes are prepared in an aprotic solvent, the reduction reaction for the remaining serotypes is carried out in an aqueous solvent (e.g., selected from PBS (phosphate buffered saline), MES (2-( N -morpholino) ethanesulfonic acid), HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), bis-tris, ADA (N-(2-acetamido)iminodiacetic acid), PIPES (piperazine-N,N′-bis(2 -ethanesulfonic acid)), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), DIPSO (3-bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazine-N-(2 -hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), bicine or HEPB, at pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5).

In some embodiments, the glycoconjugates of the present invention comprise a polysaccharide having a molecular weight between 10 kDa and 10,000 kDa. In other such embodiments, the polysaccharide has a molecular weight between 25 kDa and 5000 kDa. In other such embodiments, the polysaccharide has a molecular weight between 50 kDa and 1000 kDa. In other such embodiments, the polysaccharide has a molecular weight between 70 kD and 900 kD. In other such embodiments, the polysaccharide has a molecular weight between 100 kD and 800 kD. In other such embodiments, the polysaccharide has a molecular weight between 200 kD and 600 kD. In other such embodiments, the polysaccharide 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 - 1000 kDa; 150 kDa - 900 kDa; 150 kDa - 800 kDa; 150 kDa - 700 kDa; 150 kDa - 600 kDa; 150 kDa - 500 kDa; 150 kDa - 400 kDa; 150 kDa - 300 kDa; 200 kDa - 1000 kDa; 200 kDa - 900 kDa; 200 kDa - 800 kDa; 200 kDa - 700 kDa; 200 kDa - 600 kDa; 200 kDa - 500 kDa; 200 kDa - 400 kDa; 200 kDa - 300; 250 kDa - 1000 kDa; 250 kDa - 900 kDa; 250 kDa - 800 kDa; 250 kDa - 700 kDa; 250 kDa - 600 kDa; 250 kDa - 500 kDa; 250 kDa - 400 kDa; 250 kDa - 350 kDa; 300 kDa - 1000 kDa; 300 kDa - 900 kDa; 300 kDa - 800 kDa; 300 kDa - 700 kDa; 300 kDa - 600 kDa; 300 kDa - 500 kDa; 300 kDa - 400 kDa; 400 kDa - 1000 kDa; 400 kDa - 900 kDa; 400 kDa - 800 kDa; 400 kDa - 700 kDa; 400 kDa - 600 kDa; or 500 kDa - 600 kDa.

In specific embodiments, the conjugation reaction is carried out by reductive amination, where nickel is used to make the conjugation reaction more efficient and to help remove free cyanide. Transition metals are known to form stable complexes with cyanide and are known to improve reductive methylation of protein amino groups using formaldehyde and sodium cyanoborohydride (S Gidley et al ., Biochem J. 1982, 203: 331-334; Jentoft et al . Anal Biochem 1980, 106: 186-190). By incorporating residual, inhibitory cyanide into the complex, nickel addition increases protein utilization during conjugation and results in larger, potentially more immunogenic conjugates.

Suitable alternative chemistries include activation of the saccharide using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form the cyanate ester. The activated saccharide can thus be attached directly or via a spacer (linker) group to the amino group on the carrier protein. For example, the spacer may be a cystamine or cysteamine to provide a thiolated polysaccharide that can be attached to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using GMBS) or a haloacetylated carrier protein (e.g., using iodoacetimide [eg ethyliodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB or SIA or SBAP). Preferably, the cyanate ester (optionally produced via CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized saccharide is conjugated to the carrier protein using carbodiimide chemistry (e.g., EDAC or EDC) via the carboxyl group on the carrier protein. Such conjugates are described in International Patent Application Publication No. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu et al ., 1983, Infect. Immunity 40:245-256.

Other suitable conjugation methods use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, S-NHS, EDC, TSTU. Many of them are described in International Patent Application Publication No. WO 98/42721. The conjugation may include a carbonyl linker which may be formed by the reaction of the 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 reaction with the protein to form a carbamate bond. This may include reducing the anomeric end to a primary hydroxyl group, optionally protecting/deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

After conjugation (reduction reactions and optionally capping or quenching reactions), glycoconjugates can be purified (enriched relative to the amount of polysaccharide-protein conjugate) by a variety of methods known to the person skilled in the art. These methods include dialysis, concentration/diafiltration methods, flow-through filtration, ultrafiltration, precipitation/elution, column chromatography (ion exchange chromatography, multiple mode ion exchange chromatography, DEAE or hydrophobic interaction chromatography) and depth filtration. See, for example, US Patent No. No. 6,146,902. In one embodiment, the glycoconjugates are purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

One way to characterize the glycoconjugates of the invention is by the number of lysine residues in the carrier protein (eg, CRM197) that become conjugated to the saccharide, which can be characterized as a range of lysine residues conjugated (degree of conjugation). Evidence of a lysine modification of the carrier protein, due to covalent bonds with polysaccharides, can be obtained by analysis of amino acids using conventional methods known to the person skilled in the art. The conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to form the conjugate materials. In a preferred embodiment, the degree of conjugation of the glycoconjugate of the invention is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15 , between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12 , between 10 and 15, or between 10 and 12. In one embodiment, the degree of conjugation of the glycoconjugate 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 glycoconjugate of the invention is between 4 and 7. In some such embodiments, the carrier protein is CRM197.

The glycoconjugates of the invention can also be characterized by the ratio (w/w) of saccharide to carrier protein. In some embodiments, the ratio of polysaccharide to carrier protein in the glycoconjugate (w/w) is between 0.5 and 3.0 (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 between 0.5 and 2.0, between 0.5 and 1.5, between 0.8 and 1.2, between 0.5 and 1.0, between 1.0 and 1.5, or between 1.0 and 2.0. In further embodiments, the ratio of saccharide to carrier protein (w/w) is between 0.8 and 1.2. In a preferred embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1. In some such embodiments, the carrier protein is CRM197. The glycoconjugates and immunogenic compositions of the invention may contain a free saccharide that is not covalently conjugated to the carrier protein but is nonetheless present in the glycoconjugate composition. The free saccharide may be non-covalently associated with (ie, non-covalently associated with, adsorbed to, or incorporated into or with the glycoconjugate).

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

Multivalent vaccines based on polysaccharide-protein conjugate

In specific embodiments, the multivalent polysaccharide vaccines comprise unconjugated polysaccharides or polysaccharide-protein conjugates from Streptococcus pneumoniae serotype 31 and capsular polysaccharides from one or more of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 28A, 33F, 34, 35A, 35B, 35F and 38, in the form of free polysaccharides, a polysaccharide-protein conjugate component, or a combination thereof, to produce a multivalent pneumococcal vaccine. In specific embodiments, the immunogenic composition comprises, consists primarily of, or consists of capsular polysaccharides from S. pneumoniae serotypes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 individually conjugated to one or more carrier proteins. Preferably, saccharides from a particular serotype are not conjugated to more than one carrier protein.

After purification of the individual glycoconjugates, their compounds are obtained to formulate the immunogenic composition of the present invention. These pneumococcal conjugates are prepared by separate methods and the bulk preparation is formulated into a separate dosage form.

Pharmaceutical/vaccine compositions

In addition, the present invention relates to compositions, including pharmaceutical, immunogenic and vaccine compositions, containing, essentially consisting of, or alternatively consisting of, any of the combinations of S. pneumoniae polysaccharide serotypes described above, together with a pharmaceutically acceptable carrier and adjuvant.

The preparation of the polysaccharide-protein conjugate formulations of the present invention can be carried out using methods known in the art. For example, formulations of individual pneumococcal conjugates can be prepared using a physiologically acceptable carrier to form the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

In a preferred embodiment, the vaccine composition is formulated in L-histidine buffer with sodium chloride.

As defined herein, an "adjuvant" is a substance that serves to increase the immunogenicity of an immunogenic composition of the invention. An immune adjuvant may enhance an immune response to an antigen that is weakly immunogenic when administered alone, e.g., does not or induces weak antibody titers or a cell-mediated immune response, may increase antibody titers against the antigen, and/or reduce the dose of antigen effective to achieve an immune response at the individual. Thus, adjuvants are often administered to boost the immune response and are well known to those skilled in the art. Suitable adjuvants to enhance the effectiveness of the composition include, but are not limited to:

(1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (in the presence or absence of other specific immunostimulatory agents such as muramyl peptides (defined below) or bacterial cell wall components), such as (a) MF59 (International Patent Application Publication No. WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as a model 110Y microfluidizer (Microfluidics , Newton, MA), (b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic L121 block copolymer, and thr-MDP, either microfluidized into a submicron emulsion or shaken to form an emulsion with a coarser particle size, (c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O -deacylated monophosphoryl lipid A (MPL™) as described in US Patent No. 4912094, trehalose dimycolate (TDM) and cell wall scaffold (CWS), preferably MPL+CWS (Detox™); and (d) montanide ISA;

(3) saponin adjuvants such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, MA) (see e.g. US Pat. formed by a combination of cholesterol, saponin, phospholipid and amphipathic proteins) and ^Iscomatrix® (having basically the same structure as ISCOM, but without the protein);

(4) bacterial lipopolysaccharides, synthetic lipid A analogs such as aminoalkylglucosamine phosphate (AGP) compounds, or derivatives or analogs thereof, which are available from Corixa and which are described in US Patent No. 6113918; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl-2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3 -tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside, also known as 529 (formerly known as RC529), which is formulated in aqueous form or as a stable emulsion;

(5) synthetic polynucleotides such as oligonucleotides containing CpG motif(s) (US Patent No. 6207646);

(6) cytokines such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc. .), interferons (e.g. interferon gamma), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and etc.; And

(7) complement components such as complement component trimer C3d.

In another embodiment, the adjuvant is a mixture of 2, 3 or more of the above adjuvants, eg SBAS2 (oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycero -3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In specific embodiments, the adjuvant is an aluminum salt. The aluminum salt adjuvant may be present in an alum-precipitated or alum-adsorbed vaccine. Aluminum salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in Immunology 143:489-493). The aluminum salt includes, but is not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, ^Alhydrogel® , Superfos, ^Amphogel® , aluminum(III) hydroxide, aluminum hydroxyphosphate sulfate (phosphate based adjuvant). alumina (APA)), amorphous alumina, trihydrated alumina or trihydroxyaluminum.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA is made by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After the mixing process, the material is subjected to size reduction using a high shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered against physiological saline and steam sterilized.

In specific embodiments, commercially available Al(OH) ₃ (eg, ^Alhydrogel® or Superfos from Denmark/Accurate Chemical and Scientific Co., Westbury, NY) is used to adsorb proteins. Protein adsorption is dependent, in another embodiment, on the pI (isoelectric pH point) of the protein and the pH of the medium. A protein with a low pI is adsorbed on a positively charged aluminum ion more strongly than a protein with a high pI. Aluminum salts may form a depot of Ag that slowly releases it over a period of 2-3 weeks, may be involved in non-specific macrophage and complement activation, and/or stimulate the innate immune mechanism (possibly through stimulation of uric acid production). See, for example, Lambrecht et al ., 2009, Curr Opin Immunol 21:23.

Monovalent bulk aqueous conjugates are generally mixed and diluted. After dilution, the batch is sterilized by filtration. Aluminum phosphate adjuvant is added aseptically to a target final concentration of 4 µg/mL for all S. pneumoniae serotypes except for serotype 6B, which is diluted to a target concentration of 8 µg/mL, and a final aluminum concentration of 250 µg/mL. The formulation of the adjuvanted batch can be filled into ampoules or syringes.

In specific embodiments, the adjuvant is a CpG-containing nucleotide sequence, for example, a CpG-containing oligonucleotide, in particular a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN 1826, which can be obtained from the Coley Pharmaceutical Group.

"CpG-containing nucleotide", "CpG-containing oligonucleotide", "CpG oligonucleotide" and similar terms refer to a 6-50 nucleotide long nucleotide molecule containing an unmethylated CpG motif. See, for example, Wang et al ., 2003, Vaccine 21:4297. In another embodiment, any other definition of these terms accepted in the art is contemplated. CpG-containing oligonucleotides include modified oligonucleotides using any synthetic internucleoside linkages, a modified base and/or a modified sugar.

Methods for using CpG oligonucleotides are well known in the art and are described, for example, in Sur et al ., 1999, J Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al ., 2006, Crit Rev Ther Drug Carrier Syst. 23:89-110.

Introduction / dosing

The compositions and compositions of the present invention can be used to protect or treat a person susceptible to an infection, such as pneumococcal infection, by administering a vaccine by systemic or mucosal route. In one embodiment, the present invention relates to a method of inducing an immune response to a S. pneumoniae capsular polysaccharide conjugate comprising administering to a human an immunologically effective amount of an immunogenic composition of the present invention. In another embodiment, the present invention relates to a method of vaccinating a human against pneumococcal disease, comprising the step of administering to a human an immunologically effective amount of an immunogenic composition of the present invention.

The optimal amounts of components for a particular vaccine can be determined through standard studies, including the observation of appropriate immune responses in subjects. For example, in another embodiment, the human vaccination dose is determined by extrapolating animal data to human data. In another embodiment, the dose is determined empirically.

An "effective amount" of a composition of the invention refers to the dose required to induce the production of antibodies significantly reducing the likelihood or severity of infection by a microorganism, eg S. pneumoniae , during subsequent infection.

The methods of the invention can be used to prevent and/or reduce primary clinical syndromes caused by microorganisms such as S. pneumoniae , including both invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

Administration of the compositions of the invention may include one or more of: injection by intramuscular, intraperitoneal, intradermal, or subcutaneous routes; or mucosal administration to the oral/alimentary, respiratory or genitourinary tracts. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (because nasopharyngeal carriage of pneumococci can be more effectively prevented, thus alleviating infection at its earliest stage).

The amount of conjugates in each vaccine dose is chosen as the amount that induces an immunoprotective response without significant adverse effects. This amount can be changed depending on the pneumococcal serotype. Typically, for polysaccharide-based conjugates, each dose may contain 0.1-100 µg of each polysaccharide, in particular 0.1-10 µg, and more specifically 1-5 µg. For example, each dose may contain 100, 150, 200, 250, 300, 400, 500, or 750 ng, or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90 or 100 micrograms of each polysaccharide.

The optimal amounts of components for a particular vaccine can be determined using standard studies, including the observation of appropriate immune responses in subjects. For example, in another embodiment, the human vaccination dose is determined by extrapolating animal data to human data. In another embodiment, the dose is determined empirically.

In one embodiment, the aluminum salt dose is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700 micrograms, or 1, 1.2, 1.5, 2 , 3.5 mg or more. In another embodiment, the dose of aluminum salt described above is present per μg of the recombinant protein.

Typically, a formulation of each 0.5 ml dose is prepared containing: 2 μg of each S. pneumoniae polysaccharide except for the serotype 6B polysaccharide at 4 μg; approximately 32 μg CRM197 carrier protein (eg, 32 μg ± 5 μg, ± 3 μg, ± 2 μg, or ± 1 μg); adjuvant 0.125 mg elemental aluminum (0.5 mg aluminum phosphate); and sodium chloride and L-histidine buffer. The sodium chloride concentration is approximately 150 mM (for example, 150 mM ± 25 mM, ± 20 mM, ± 15 mM, ± 10 mM, or ± 5 mM), and approximately 20 mM (for example, 20 mM ± 5 mM, ± 2, 5 mM, ± 2 mM, ± 1 mM, or ± 0.5 mM) L-histidine buffer.

In accordance with any of the methods of the present invention, and in one embodiment, the subject is a human. In specific embodiments, the human patient is an infant (less than 1 year of age), a preschool child (about 12-24 months old), or a preschool child (about 2-5 years old). In other embodiments, the human patient is an elderly patient (>65 years of age). The compositions of this invention are also suitable for use in older children, adolescents and adults (eg aged 18-45 or 18-65).

In one embodiment of the methods of the present invention, the composition of the present invention is administered in the form of a single inoculation. In another embodiment, the composition is administered two times, three times, or four times, or more, at adequate intervals. For example, the composition can be administered at intervals of 1, 2, 3, 4, 5, or 6 months, or any combination thereof. You can follow the immunization schedule developed for pneumococcal vaccines. For example, a common schedule for infants and preschool children against invasive disease caused by S. pneumoniae is administration at 2, 4, 6, and 12-15 months of age. Thus, in a preferred embodiment, the composition is administered in a series of 4 doses at 2, 4, 6 and 12-15 months of age.

Compositions of the present invention may also include one or more proteins from S. pneumoniae . Examples of S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publication Nos. WO 02/083855 and WO 02/053761.

Lineups

The compositions of the present invention may be administered to a subject by one or more routes known to those skilled in the art, for example, parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally, and formulated as appropriate.

In one embodiment, the compositions of the present invention are administered by epidermal injection, intramuscular injection, intravenous, intra-arterial, subcutaneous injection, or mucosal injection of a liquid preparation into the respiratory tract. Liquid formulations for injection include solutions and the like.

The composition of the present invention may be in the form of single dose ampoules, multi-dose vials or pre-filled syringes.

In another embodiment, the compositions of the present invention are administered orally, and thus formulated in a form suitable for oral administration, ie, in the form of a solid or liquid preparation. Solid oral formulations include tablets, capsules, pills, granules, dragees, and the like. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils, and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish oil, other marine animal fat or lipid from milk or eggs.

The pharmaceutical composition may be isotonic, hypertonic or hypotonic. However, it is often preferred that the pharmaceutical composition for infusion or injection is substantially isotonic when administered. Thus, for storage, the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it may be diluted to an isotonic solution prior to administration.

The tonicity agent can be an ionic tonicity agent such as a salt or a non-ionic tonicity agent such as a carbohydrate. Non-limiting examples of ionic tonicity agents include NaCl, CaCl ₂ , KCl and MgCl ₂ . Non-limiting examples of non-ionic tonicity agents include sucrose, trehalose, mannitol, sorbitol, and glycerin.

It is also preferred that at least one pharmaceutically acceptable additive is a buffer. For some purposes, for example, when the pharmaceutical composition is intended for infusion or injection, it is often desirable that the composition contains a buffer capable of buffering the solution at a pH in the range of 4-10, eg 5-9, eg 6-8.

The buffer may, for example, be selected from the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate and triethanolamine buffers.

The buffer may also be selected from, for example, USP-acceptable buffers for parenteral use, in particular when the pharmaceutical composition is intended for parenteral use. For example, the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic acid; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric acids; polybasic acids such as citric and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine and Tris.

Carriers for parenteral administration (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, and fixed oils. Carriers for intravenous administration include fluids and nutritional supplements, electrolyte supplements such as those based on dextrose Ringer's solution, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Generally, water, saline, aqueous solutions of dextrose and related sugars, glycols such as propylene glycols or polyethylene glycol, polysorbate 80 (PS-80), polysorbate 20 (PS-20), and poloxamer 188 (P188) are the preferred liquid carriers. in particular for injectable solutions. Examples of oils are oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish oil, other marine animal fat or lipid from milk or eggs.

The compositions may also contain a surfactant. Preferred surfactants include, but are not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tween), especially PS-20 and PS-80; ethylene oxide (EO), propylene oxide (PO) and/or butylene oxide (BO) copolymers sold under the trade name DOWFAX™, such as EO/PO linear block copolymers; octoxynols, which can be variable in the number of ethoxy(oxy-1,2-ethanediyl) repeating groups, with octoxynol-9 (triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates such as the Tergitol™ NP series; polyoxyethylene fatty esters derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPAN) such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for inclusion in the emulsion are PS-20 or PS-80.

Mixtures of surfactants can be used, for example mixtures of PS-80/Span 85. Also suitable is a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100). Another combination that can be used includes laureth 9 plus polyoxyethylene sorbitan ester and/or octoxynol.

Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as PS-80) 0.01-1% w/v, in particular about 0.1% w/v; octyl or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001-0.1% w/v, in particular 0.005-0.02% w/v; polyoxyethylene ethers (such as laureth 9) 0.1-20% wt./about., preferably 0.1-10% wt./about. and in particular, 0.1-1% wt./about. or approximately 0.5% wt./about.

In specific embodiments, the implementation, the composition mainly consists of L-histidine (20 mm), saline (150 mm) and 0.2% wt./about. PS-20 at pH 5.8 with 250 µg/ml APA (Aluminum Phosphate Adjuvant). PS-20 may be in the range of 0.005-0.1% w/v, where the presence of PS-20 or PS-80 in the formulation controls aggregation during simulated manufacturing and transport using primary packaging. The method consists of combining a mixture of up to 44 S. pneumoniae polysaccharide serotypes in L-histidine, sodium chloride and PS-20, then combining the mixed material with APA and sodium chloride in the presence or absence of antimicrobial preservatives.

The choice of surfactant may need to be optimized for different drug products and drug substances. For multivalent vaccines containing 15 or more S. pneumoniae polysaccharide serotypes, PS-20 and P188 are preferred. The choice of chemical reactions used to obtain the conjugate can also affect the stabilization of the composition. In particular, as illustrated below, pneumococcal polysaccharide-protein conjugates prepared in an aqueous or DMSO-based solvent and combined into a multivalent formulation show significant differences in stability, depending on the specific surfactant systems used to formulate the formulation.

For the formulations described herein, the poloxamer typically has a molecular weight in the range of 1100 Da to 17400 Da, 7500 Da to 15000 Da, or 7500 Da to 10000 Da. The poloxamer may be selected from poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the compositions of the invention is 0.001 to 5% w/v, or 0.025 to 1% w/v. The surfactant system containing the poloxamer must additionally contain a polyol. In specific aspects, the polyol is propylene glycol and is present at a final concentration of 1 to 20% w/v. In specific aspects, the polyol is a polyethylene glycol 400 and is present at a final concentration of from 1 to 20% wt./about.

Suitable formulation polyols are polymeric polyols, in particular polyester diols, including but not limited to propylene glycol and polyethylene glycol, polyethylene glycol monomethyl ethers. Propylene glycol is available in the monomer molecular weight range from ~425 Da to ~2700 Da. Polyethylene glycol and polyethylene glycol monomethyl ethers are also available in the molecular weight range from ~200 Da to ~35,000 Da including, but not limited to, PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350 and PEG MME 4000. The preferred polyethylene glycol is polyethylene glycol 400. The final concentration of the polyol in the compositions can be 1-20% wt./about. or 6-20% wt./about.

The formulation also contains pH buffered saline. The buffer may, for example, be selected from the group consisting of Tris buffer, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate, HEPES (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-( N -morpholino)ethanesulfonic acid) and triethanolamine. The buffer is capable of buffering the solution to a pH in the range of 4-10, 5.2-7.5, or 5.8-7.0. In specific aspects, the buffer is selected from the group consisting of phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate, or citrate. The buffer may also be selected from, for example, USP-acceptable buffers for parenteral use, in particular when the pharmaceutical composition is intended for parenteral use. Buffer concentrations may range from 1 mM to 50 mM, or from 5 mM to 50 mM. In specific aspects, the buffer is L-histidine at a final concentration of 5 mM-50 mM, or succinate at a final concentration of 1 mM-10 mM. In specific aspects, L-histidine is present at a final concentration of 20 mM ± 2 mM.

While a saline solution (ie, a solution containing NaCl) is preferred, other salts suitable for formulation include, but are not limited to, CaCl ₂ , KCl and MgCl ₂ , and combinations thereof. Non-ionic tonicity agents, including but not limited to sucrose, trehalose, mannitol, sorbitol, and glycerin, may be used in place of salt. Suitable salt concentration ranges include, but are not limited to, 25 mM to 500 mM, or 40 mM to 170 mM. In one aspect, the saline solution is NaCl, optionally present at a concentration of 20 mM to 170 mM.

In a preferred embodiment, the formulations contain L-histidine buffer with sodium chloride.

In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the agent can be administered using intravenous infusion, transdermal patch, liposomes, or other modes of administration. In another embodiment, polymeric materials are used; for example, in microspheres or an implant.

Compositions of the present invention may also include one or more proteins from S. pneumoniae . Examples of S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publication No. WO 02/083855 and WO 02/053761.

Analytical methods

Molecular weight and concentration analysis of conjugates using HPSEC/UV/MALS/RI analysis

Conjugate samples are injected and separated by high performance size exclusion chromatography (HPSEC). Detection is carried out using a series of ultraviolet light (UV), multi-angle light scattering (MALS) and refractive index (RI) detectors. The protein concentration is calculated from UV280 using the extinction coefficient. The polysaccharide concentration is deconvoluted from the RI signal (contributed by both the protein and the polysaccharide) using the dn/dc factors, which are the change in the refractive index of the solution as the solute concentration changes, expressed in ml/g. The average molecular weight of the samples was calculated using Astra software (Wyatt Technology Corporation, Santa Barbara, CA) using the measured concentration and light scattering information for the entire sample peak. There are many forms of average molecular weights for polydisperse molecules. For example, the number average molecular weight Mn, the weight average molecular weight Mw, and the z-average molecular weight Mz (Molecules, 2015, 20:10313-10341). Unless otherwise indicated, the term "molecular weight", as used throughout this description, is a weight average molecular weight.

Determination of the use of lysine in a conjugated protein as an indicator of the number of covalent bonds between the polysaccharide and the carrier protein

The AccQ-Tag Amino Acid (AAA) assay from Waters is used to measure the degree of conjugation in conjugate samples. Samples are hydrolyzed using gas phase acid hydrolysis in an Eldex workstation to cleave carrier proteins into their amino acid components. Free amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC). The derivatized samples are then analyzed using UPLC with UV detection on a C18 column. The average protein concentration is obtained using representative amino acids other than lysine. The use of lysine during conjugation (ie, the loss of lysine) is determined by the difference between the average measured amount of lysine in the conjugate and the expected amount of lysine in the original protein.

Free polysaccharide a testing

Free polysaccharide (ie, polysaccharide not conjugated to CRM197) in the conjugate is measured by first precipitating free protein and conjugates using deoxycholate (DOC) and hydrochloric acid. The precipitates are then filtered off and the filtrates are analyzed for free polysaccharide concentration by HPSEC/UV/MALS/RI. Free polysaccharide is calculated as the percentage of total polysaccharide measured by HPSEC/UV/MALS/RI.

Free protein testing

Free polysaccharide, polysaccharide-CRM197 conjugate and free CRM197 in conjugate samples are separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. Briefly, samples are mixed with MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3 and separated in pre-prepared uncoated fused silica capillaries. Separation is monitored at 200 nm and free CRM197 is quantified using a CRM197 standard curve. Free protein results are reported as a percentage of total protein as determined by the HPSEC/UV/MALS/RI method.

While various embodiments of the invention have been described with reference to the accompanying description and drawings, it should be understood that the invention is not limited to these exact embodiments, and that various changes and modifications may be made by a person skilled in the art without deviating from the scope or content of the invention as defined. in the appended claims.

The following examples illustrate but do not limit the invention.

EXAMPLES

EXAMPLE 1 Preparation of S. pneumoniae capsular polysaccharides

Methods for culturing pneumococci are well known in the art. See, for example, Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. EP 0 497 524 B1. The process described below basically follows the method described in European Patent No. EP 0 497 524 B1, and is generally applicable to all pneumococcal serotypes except specifically modified ones.

Pneumococcal serotype 31 strains were obtained from the Centers for Disease Control and Prevention (Atlanta, GA). If necessary, subtypes can be differentiated based on the swelling response using specific antisera. See, for example, US Patent No. No. 5,847,112. The obtained isolates were further subjected to clonal isolation by serial plating in two stages on agar plates consisting of an animal-free medium containing soy peptone, yeast extract and glucose without hemin. Clonal isolates for each serotype were further expanded in liquid culture using animal-free media containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare preliminary master cell banks.

The preparation of each pneumococcal polysaccharide serotype consisted of cell expansion and production by batch fermentation, followed by chemical inactivation prior to downstream purification. The thawed cell bank vial for each serotype was expanded using a shake flask or culture bottle containing pre-sterilized animal-free growth media containing soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate and glucose. The cell expansion culture was grown in a sealed shake flask or bottle to minimize gas exchange, with temperature control and shaking. After reaching the specified culture density, as measured by absorbance at 600 nm, a portion of the cell expansion culture was transferred to a production fermenter containing pre-sterilized animal-free growth media containing soy peptone or soy peptone ultrafiltrate, yeast extract or ultrafiltrate. yeast extract, sodium chloride, potassium phosphate and glucose. Temperature, pH, pressure and shaking were controlled. The flow of air was also controlled as bubbling was not used.

Batch fermentation was stopped by adding a chemical inactivator, phenol, when the glucose was nearly depleted. Pure phenol was added to a final concentration of 0.8-1.2% to inactivate the cells and release the capsular polysaccharide from the cell wall. Primary inactivation takes place over a period of time inside the fermenter, where temperature and shaking must continue to be controlled. After primary inactivation, the batch was transferred to another vessel where it was maintained for an additional specified time at controlled temperature and shaking for complete inactivation. This was confirmed either by microorganism sieving methods or by checking the phenol concentration and the indicated time. The inactivated broth was then purified.

Purification Ps

Pneumococcal polysaccharide purification consists of several centrifugation, depth filtration, concentration/diafiltration and precipitation steps. All methods were performed at room temperature unless otherwise noted.

The inactivated S. pneumoniae culture broth from the fermenter was flocculated with a cationic polymer (such as BPA-1000, Petrolite "Tretolite" and "Spectrum 8160", and poly(ethyleneimine), "Millipore pDADMAC"). Cationic polymers bind to contaminating proteins, nucleic acids and cell debris. After a flocculation step and a holding period, the flocculated solids were removed by centrifugation and multiple depth filtration steps. The clarified broth was concentrated and subjected to diafiltration using a filter with MWCO (cut off by molecular weight) 100 kDa - 500 kDa. Diafiltration was carried out using Tris buffer, MgCl ₂ and sodium phosphate buffer. Diafiltration removed residual nucleic acid and protein.

Additional removal of contaminants was carried out by re-precipitation of the polysaccharide in sodium acetate and phenol with denatured alcohol and/or isopropanol. During the phenol precipitation step, sodium acetate in sodium phosphate buffered saline and phenol (thinned phenols or solid phenols) were loaded into the retentate after diafiltration. Then alcohol fractionation of the polysaccharide was carried out in two stages. In the first step, a low percentage of alcohol was added to the formulation to precipitate cell debris and other unwanted contaminants while the crude polysaccharide remained in solution. Contaminants were removed by centrifugation followed by a depth filtration step. The polysaccharide was then isolated from solution by adding additional isopropanol or denatured alcohol to the batch. The precipitated polysaccharide precipitate was isolated by centrifugation, pulverized and dried in powder form, and stored frozen at -70°C.

EXAMPLE 2 Polysaccharide Structure Analyzes by NMR

The method for determining the structure of a polysaccharide involved a multi-step process essentially as described in Abeygunawardana et al., Determination of the Chemical Structure of Complex Polysaccharides by Heteronuclear NMR Spectroscopy in Advances in Biophysical Chemistry 1993, Vol 3, pages 199-249, JAI Press Inc. The purified polysaccharides were examined using standard 1D and 2D NMR methods. Since polysaccharides from S. pneumoniae serotype 31 were identified as containing O-acetyl, a detailed analysis was performed for the de-O-acetylated polysaccharide (O-acetate groups were removed using basic hydrolysis). Finally, the polysaccharides were examined for the presence of phosphate using ³¹ P NMR.

Determination of monosaccharide residues was performed by ¹ H- ¹ H COSY, two-quantum filtering homonuclear COSY and total correlation spectroscopy (TOCSY). ¹³ C chemical shifts were assessed by heteronuclear single quantum interaction (HSQC) spectroscopy and HSQC-TOCSY combination. Multiplicity corrected HSQC was used to distinguish between methylene and methine groups. Relationships between residues were determined by a combination of HMBC and NOESY spectroscopy. The anomeric configuration of the residues was determined from the chemical shifts of the anomeric proton and carbon, ³ J _H1,H2 and ¹ J _H1,C1 . The anomeric configuration of the furanose rings is difficult to assess from the ³ J _H1,H2 and ¹ J _H1,C1 values, thus long-range compounds of ring protons from the anomeric carbon were used for this determination.

In the S. pneumoniae serotype 31 polysaccharide, three separate O-methyl resonance signals were observed in the ¹ H NMR spectrum, indicating that the polysaccharide was O-acylated at three separate sites. The localization of O-acetyl groups on the purified polysaccharide was determined by analyzing chemical shift changes due to the effect of O-acetylation. A large change in the chemical shift ¹ H towards the weak field by 0.8-0.5 ppm. is an indicator of O-acetyl substitution. Carbonyls were assigned to their respective residues by CH long range heteronuclear multiply connected correlation spectroscopy (HMBC) and ¹³ C NMR spectroscopy.

Based on the NMR data in figures 2-5, it was determined that the structure of the polysaccharide of S. pneumoniae serotype 31 is as follows:

where n is the number of repeat units that make up the polysaccharide. See also figure 1.

Sugar residues include rhamnose (Rha), galactose (Gal), glucose (Glc) and Glc p A (glucuronic acid). One of the galactose residues is in the form of furanose.

The italicized letters ( p and f ) refer to pyranose (a closed ring of six atoms) and furanose (a closed ring of five atoms).

α and β refer to the configuration of the proton associated with the anomeric carbon in the sugar unit. The anomeric carbon is always number 1 when referring to the carbon atoms in the sugar unit (usually from 1 to 6). α denotes that the anomeric proton is in the equatorial position in the 3D structure. β denotes that the anomeric proton is in the axial position.

The numbers linked by arrows indicate how the individual sugar units are linked to each other. For example, the nomenclature α-Rha p -(1→3)-α-Glc p - means that carbon atom number 1 of rhamnose is bonded to carbon atom number 3 of glucose ( p denotes that both sugars are pyranose rings).

An O-acetyl (OAc) group is present on two of the sugar residues. O-acetyl is found in all three positions in approximately 90%.

EXAMPLE 3 Conjugation of S. pneumoniae serotype 31 polysaccharide to CRM197 using reductive amination in dimethyl sulfoxide

The polysaccharide was dissolved, size-adjusted to the target molecular weight, chemically activated and subjected to buffer exchange via ultrafiltration. The activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in dimethyl sulfoxide (DMSO). The solutions of the re-dissolved polysaccharide and CRM197 were then combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before final filtration through a 0.2 micron filter. Several process parameters, such as pH, temperature, concentration and time, were controlled at each stage to obtain conjugates with the desired features.

Size reduction and oxidation of the polysaccharide

Purified pneumococcal capsular Ps powder was dissolved in water and filtered through a 0.45 micron filter. The size of the dissolved polysaccharide was reduced either by acid hydrolysis or by homogenization. Acid hydrolysis was carried out by adding acetic acid up to 200 mM, incubation at 90° C. for 30 minutes, then neutralizing by adding cold potassium phosphate buffer, pH 7, up to 400 mM. For homogenization, the pressure and the number of passes through the homogenizer were controlled to 400 bar (40 MPa)/5 passes.

The reduced size polysaccharide was concentrated and diafiltered against water using a downstream ultrafiltration membrane with NMWCO 5.

The polysaccharide solution was then adjusted to 22° C. and pH 5 using sodium acetate buffer to minimize polysaccharide size reduction due to activation. Polysaccharide activation was started by adding 100 mM sodium metaperiodate solution. The sodium metaperiodate added was 0.16 mol sodium metaperiodate per mol polysaccharide repeating unit to achieve the target level of polysaccharide activation (mol aldehyde per mol polysaccharide repeating unit). The oxidation reaction was continued for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, followed by diafiltration against water using a downstream ultrafiltration membrane with 5 kDa NMWCO. Ultrafiltration was carried out at 2-8°C.

Conjugation of polysaccharide with CRM197

Purified CRM197, obtained by expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1), was diafiltered against 2 mM phosphate buffer, pH 7.0, using a flow ultrafiltration membrane along the flow with NMWCO 5 kDa and filtered through the filter 0.2 micron.

Received the composition of activated polysaccharides for lyophilization at 6 mg Ps/ml with a concentration of sucrose 5% wt./about. Received the composition of CRM197 for lyophilization at 6 mg Pr/ml with a concentration of sucrose 1% wt./about.

Solutions of Ps and CRM197 formulations were individually lyophilized. Lyophilized Ps and CRM197 materials were redissolved individually in equal volumes of DMSO. Polysaccharide and CRM197 solutions were mixed to achieve a polysaccharide concentration of 2.8-4.0 g Ps/l (grams of polysaccharide/liter) and a weight ratio of polysaccharide and CRM197 of 1.5. The weight ratio was chosen to control the ratio of polysaccharide and CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeating unit) was added and conjugation continued for 1 hour at 22°C.

Recovery using sodium borohydride

Sodium borohydride (2 mol per mol of polysaccharide repeating unit) was added after the conjugation reaction and incubated for 1 hour at 22°C. The batch was diluted in 150 mm sodium chloride with approximately 0.025% (wt./about.) polysorbate 20, at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. For some batches, the batch was concentrated and diafiltered at approximately 4° C. against 150 mM sodium chloride, 25 mM potassium phosphate pH 7 using a downstream ultrafiltration membrane with 30 kDa NMWCO.

Final filtration and product storage

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, at 4° C. using a downstream ultrafiltration membrane with NMWCO 300 kDa .

The batch of retentate was filtered through a 0.2 micron filter, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20, aliquoted and frozen at ≤ − 60°C.

Table 1 shows features of S. pneumoniae serotype 31 polysaccharide conjugate made in DMSO

Table 1. Signs of S. pneumoniae serotype 31 polysaccharide conjugate after conjugation in DMSO

Mw of oxidized Ps Mw conjugate Ps: Pr Lysine utilization (mol/mol CRM197) Free Ps / Shared Ps Free Protein/Total Protein 119 kDa 2999 kDa 1.15 9.5 0.7% <1%

EXAMPLE 4 Preparation of Monovalent Conjugate Formulations

Pneumococcal polysaccharide-CRM197 conjugates were prepared as described in Example 3. The required volume of bulk conjugates required to achieve a target concentration of individual serotypes was calculated based on the batch volume and concentrations of individual bulk polysaccharides. Bulk conjugateS. pneumoniae serotype 31 was combined with vehicles, sterilized by filtration and added to APA under agitation. Final concentration of monovalent conjugate vaccineS. pneumoniae serotype 31 was 4 μg/ml (w/v PnPs) with 20 mM histidine, 150 mM NaCl, 0.2% (w/v) PS-20 and 0.250 mg/ml (w/v Al) in the form of APA.

EXAMPLE 5 Immunogenicity study of a monovalent conjugate in New Zealand albino rabbits

The immunogenicity of the monovalent conjugates was evaluated in the New Zealand albino rabbit (NZWR) model. Adult New Zealand albino rabbits (NZWR, n=3/group) intramuscularly (IM) were immunized with 0.25 ml of the respective monovalent conjugate vaccine on day 0 and day 14 (changing sides). The monovalent pneumococcal vaccine was dosed at 1 μg of PnPs ( S. pneumoniae serotype 31 polysaccharide conjugated to CRM197) with 62.5 μg of aluminum phosphate adjuvant (APA) for immunization. Sera were collected before the start of the study (before immunization) and on days 14 (after dosing 1, PD1) and 28 (after dosing 2, PD2). The NZWR was examined at least daily by a qualified animal care professional for any signs of disease or illness. The NZWR vaccine formulations appeared to be safe and well tolerated. All animal experiments were performed in strict accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR protocol has been approved by the Animal Care and Use Committees of both Merck & Co., Inc (Kenilworth, NJ) and Covance (Denver, PA).

NZWR sera were tested in ELISA assays to assess IgG immunogenicity using 1-2 mg/mL coverage of the corresponding PnPs to cover. Functional antibody was determined by opsonophagocytosis assays (OPA), based on previously described methods. See, for example, Caro-Aguilar et al ., 2017, Vaccine 35:865-72 and Burton et al ., 2006, Clin Vaccine Immunol 13(9):1004-9.

It was found that monovalent pneumococcal conjugate vaccines from S. pneumoniae serotype 31 were immunogenic in rabbits (figure 6) and led to the formation of a functional antibody that destroys the corresponding bacterial strain (figure 7).

EXAMPLE 6 Preparation of Pneumococcal Conjugate Vaccine Formulations for a Multivalence Study in Rabbits

A multivalent pneumococcal conjugate vaccine consisting of a mixture of bulk preparations of various conjugates (including S. pneumoniae serotypes 16F, 23A, 23B, 24F, and 31) was prepared using pneumococcal polysaccharide-CRM197 conjugates and formulated in 20 mM histidine, pH 5.8, and 150 mm sodium chloride, and 0.1% wt./about. polysorbate-20 (PS-20) at 4 µg/ml of each serotype to a total polysaccharide concentration of 84 µg/ml. Conjugates were obtained by individual conjugation of the CRM197 protein with pneumococcal polysaccharide (PnPs) types (including S. pneumoniae serotypes 16F, 23A, 23B, 24F and 31). The required volume of bulk conjugates required to achieve the target concentration of individual serotypes was calculated from the batch volume and the concentrations of individual bulk polysaccharides. Individual conjugates were added to a solution of histidine, sodium chloride and polysorbate-20 (PS-20) to obtain a mixture of conjugates. The formulation vessel containing the mixture of conjugates was agitated using a magnetic stirrer arm and sterile filtered into another vessel. The formulations were then filled into plastic syringes, glass syringes or vials and stored at 2-8 ^° C.

EXAMPLE 7 Immunogenicity study of a multivalent pneumococcal conjugate vaccine in New Zealand albino rabbits

Adult albino New Zealand rabbits (NZWR, n=5/group) intramuscularly (IM) were immunized with 0.5 ml of the multivalent pneumococcal conjugate vaccine described in Example 8 on day 0 and day 14 (changing sides). The pneumococcal vaccine was dosed at 2 μg of each PnPs conjugated per immunization. Sera were collected before the start of the study (before immunization) and on days 14 (after dosing 1, PD1) and 28 (after dosing 2, PD2). The NZWR was examined at least daily by a qualified animal care professional for any signs of disease or illness. The NZWR vaccine formulations appeared to be safe and well tolerated. All animal experiments were performed in strict accordance with the recommendations of the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR protocol has been approved by the Animal Care and Use Committees of both Merck & Co., Inc. and Covance (Denver, PA).

NZWR sera were assessed for IgG immunogenicity using multiplex electrochemiluminescence (ECL) assay. This assay is designed for use with rabbit sera based on the human assay described in Marchese et al . (Optimization and validation of a multiplex, electrochemiluminescence-based detection assay for the quantitation of immunoglobulin G serotype-specific antipneumococcal antibodies in human serum. Clin Vaccine Immunol . 16(3): 387-96 (2009)), using technology developed by in MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD), which uses a SULFO-TAG™ tag that emits light after electrochemical stimulation. SULFO-TAG™ labeled anti-rabbit IgG was used as a secondary antibody for testing NZWR serum samples. Functional antibody was determined by multiplex opsonophagocytosis assays (MOPA), based on previously described methods available online from the Bacterial Respiratory Pathogen Reference Laboratory at the University of Alabama at Birmingham, using Opsotiter® 3 software (UAB Research Foundation, Caro-Aguilar et al . 2017 supra, Burton et al ., 2006 supra).

Polysaccharide-protein conjugates derived from S. pneumoniae serotypes 16F, 23A, 23B, 24F, and 31 in multivalent pneumococcal conjugate vaccines were found to be immunogenic both after dosing 1 (PD1) and after dosing 2 (PD2), in rabbits (figure 8). They also led to the formation of a functional antibody that destroys bacterial strains related to the types from the vaccine (figure 9). Rabbits immunized with the 2 μg multivalent pneumococcal conjugate vaccine had significantly higher MOPA PD1 titers for the four serotypes compared to pre-immunization rabbit sera (Figure 9). Rabbits immunized with the 2 μg multivalent pneumococcal conjugate vaccine had significantly higher MOPA PD2 titers for all five serotypes compared to pre-immunization rabbit sera (Figure 9). Log-transformed data were analyzed by one-way ANOVA using Dunnett's test to determine significance.

Claims (30)

1. Polysaccharide-carrier protein conjugate, where the polysaccharide contains a repeating unit of the following structure: , where each x independently denotes a molar ratio from 0 to 1.0; wherein the carrier protein is CRM197 and further wherein the polysaccharide-carrier protein conjugate has a polysaccharide to carrier protein weight ratio of 0.5 to 2.0. 2. The polysaccharide-carrier protein conjugate of claim 1, wherein the degree of conjugation of the conjugate is between 2 and 15. 3. The polysaccharide-carrier protein conjugate of claim 1, wherein the degree of conjugation of the conjugate is between 3 and 13. 4. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide-carrier protein conjugate has a weight ratio of polysaccharide to carrier protein of 0.5 to 1.5. 5. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide-carrier protein conjugate has a polysaccharide to carrier protein weight ratio of 0.8 to 1.2. 6. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide-carrier protein conjugate contains less than about 50% non-covalently associated polysaccharide relative to the total polysaccharide. 7. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide-carrier protein conjugate contains less than about 25% non-covalently associated polysaccharide relative to the total polysaccharide. 8. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide-carrier protein conjugate contains less than about 20% non-covalently associated polysaccharide relative to the total polysaccharide. 9. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide-carrier protein conjugate contains less than about 15% non-covalently associated polysaccharide relative to the total polysaccharide. 10. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide has a molecular weight of 25 to 5,000 kDa. 11. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide has a molecular weight of 50 to 1,000 kDa. 12. The polysaccharide-carrier protein conjugate of claim 1, wherein the polysaccharide has a molecular weight of 100 to 300 kDa. 13. Conjugate polysaccharide-carrier protein according to claim 2, where the degree of conjugation is from 3 to 13; where the polysaccharide has a molecular weight of from 100 to 300 kDa; where the mass ratio of polysaccharide and carrier protein in the conjugate is from 0.5 to 1.5; wherein the polysaccharide-carrier protein conjugate contains less than about 15% non-covalently associated polysaccharide relative to the total polysaccharide; and wherein the polysaccharide-carrier protein conjugate has a molecular weight of 1,000 to 10,000 kDa. 14. Polysaccharide-carrier protein conjugate, where the polysaccharide contains a repeating unit of the following structure: , where each x independently denotes a molar ratio from 0 to 1.0; wherein the carrier protein is CRM197 and further wherein the polysaccharide-carrier protein conjugate has a molecular weight of 1,000 to 10,000 kDa. 15. The polysaccharide-carrier protein conjugate of claim 14, wherein the degree of conjugation of the conjugate is between 2 and 15. 16. The polysaccharide-carrier protein conjugate of claim 14, wherein the degree of conjugation of the conjugate is between 3 and 13. 17. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide-carrier protein conjugate has a weight ratio of polysaccharide to carrier protein of 0.5 to 2.0. 18. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide-carrier protein conjugate has a weight ratio of polysaccharide to carrier protein of 0.5 to 1.5. 19. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide-carrier protein conjugate has a weight ratio of polysaccharide to carrier protein of 0.8 to 1.2. 20. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide-carrier protein conjugate contains less than about 50% non-covalently associated polysaccharide relative to the total polysaccharide. 21. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide-carrier protein conjugate contains less than about 25% non-covalently associated polysaccharide relative to the total polysaccharide. 22. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide-carrier protein conjugate contains less than about 20% non-covalently associated polysaccharide relative to the total polysaccharide. 23. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide-carrier protein conjugate contains less than about 15% non-covalently associated polysaccharide relative to the total polysaccharide. 24. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide has a molecular weight of 25 to 5,000 kDa. 25. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide has a molecular weight of 50 to 1,000 kDa. 26. The polysaccharide-carrier protein conjugate of claim 14, wherein the polysaccharide has a molecular weight of 100 to 300 kDa.

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