Group b streptococcus polysaccharide-protein conjugates, methods for producing conjugates, immunogenic compositions comprising conjugates, and uses thereof

Abstract

To provide immunogenic polysaccharide-protein conjugates for inducing an immune response against group B streptococcus (GBS) in subjects and / or for reducing or preventing invasive GBS disease in subjects.SOLUTION: Provided are immunogenic polysaccharide-protein conjugates comprising a capsular polysaccharide (CP) from Streptococcus agalactiae and a carrier protein, the CP being selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX, and the CP having a sialic acid level of greater than about 60%. Also provided are methods of making the conjugates and immunogenic compositions comprising the conjugates, and immunogenic compositions comprising polysaccharide-protein conjugates, the conjugates comprising a CP from GBS serotype IV and at least one additional serotype.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to immunogenic polysaccharide-protein conjugates comprising capsular polysaccharide (CP) from Streptococcus agalactiae, commonly referred to as group B streptococcus (GBS), and a carrier protein, wherein the CP is selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX, and the CP has a sialic acid level of greater than about 60%. The present invention also relates to methods of making the conjugates and immunogenic compositions comprising the conjugates. The present invention also relates to immunogenic compositions comprising polysaccharide-protein conjugates, the conjugates comprising CP from GBS serotype IV and at least one additional serotype. The present invention further relates to methods for eliciting an immune response against GBS in a subject and / or for reducing or preventing invasive GBS disease in a subject using the compositions disclosed herein. The resulting antibodies can be used to treat or prevent GBS infection via passive immunotherapy. [Background technology]

[0002] Streptococcus agalactiae, also known as group B streptococci (GBS), are gram-positive polysaccharide-encapsulated organisms. They are common commensals of the human gastrointestinal and genital tracts and cause severe disease in infants and the elderly (Baker, CJ, Vaccine, 31(Suppl. 4):D3-D6 (2013)). The major risk factor for GBS infection in infants is maternal colonization (Dillon, HC, et al., J. Pediatr., 110(1):31-36 (1987)). As many as one in four women carry GBS in the rectovaginal cavity, which can infect the amniotic fluid or newborn before or during birth, causing sepsis, pneumonia and meningitis (Baker 2013; Heath, PT et al., BMJ Clin. Evid. (Online), pii:0323 (2014)). Twenty-five percent of infants who survive GBS meningitis suffer from neurological dysfunction, and 19% experience cognitive delay, cerebral palsy, blindness and hearing loss (Libster, R. et al., Pediatrics, 130(1):e8-15 2012 (2012)). GBS can also cause miscarriage and premature birth, and is associated with stillbirth (McDonald, HM et al., Infectious Diseases in Obstetrics and Gynecology, 8(5-6):220-227(2000);Randis,TM et al., The Journal of Infectious Diseases, 210(2):265-273(2014);Kessous,R. et al., J.Matern.Fetal Neonatal Med., 25(10):1983-1986(2012)).Very low birth weight infants are at much higher risk of infection, with up to 3% being infected and up to 30% mortality, even with immediate antibiotic treatment (Heath 2014).

[0003] The introduction of GBS screening and intrapartum antibiotic prophylaxis (IAP) in the United States in the late 1990s demonstrated a reduction in the rate of neonatal disease occurring within the first week of life (early onset disease [EOD]) but had no measurable impact on the rate of late onset disease (LOD) that occurs within the first 3 months of life. The rates of EOD and LOD cases in the United States are currently 0.25 and 0.27 per 1,000 live births, respectively (Centers for Disease Control and Prevention (CDC), Active Bacterial Core (ABC) Surveillance Report (2013), http: / / www.cdc.gov / abcs / reports-findings / survreports / gbs13 Following the introduction of pneumococcal conjugate vaccines for the prevention of invasive pneumococcal disease, including bacteremia and meningitis, and despite IAP for the prevention of GBS disease, GBS has become the single most common cause of neonatal sepsis (EOD) and meningitis in infants (<2 months of age) in the United States (Verani, JR et al., MMWR, 59(RR10):1-32 (2010); Thigpen, MC et al., New England Journal of Medicine, 364(21):2016-2025 (2011)). Unlike in the United States, the introduction of prevention guidelines for invasive GBS disease and IAP did not reduce the incidence of EOD in the Netherlands or in the UK (Bekker, V. et al., The Lancet Infectious Diseases, 14(11):1083-1089 (2014); Lamagni, T. L. et al., Clin. Infect. Dis., 57(5):682-688 (2013)). This lack of effect may be due to the lack of universal screening and the restriction of IAP to mothers in the highest risk groups (e.g., fever, long-standing ruptured membranes). Rates of EOD were significantly higher in countries without IAP, with a mean incidence of 0.75 per 1,000 live births (95% CI 0.58-0.89) reported (Edmond, K. M. et al., Lancet, 379(9815):547-556 (2012)).

[0004] Another population at risk for GBS disease is the elderly. Risk factors include chronic medical problems such as diabetes mellitus, cancer, heart failure, neurological and urological conditions. According to CDC ABC surveillance data, the annual US incidence rate of invasive GBS in 2013 was 0.28 / 1,000 or 12,400 cases / year in adults aged 65 years and older. This rate is similar to the attack rate of invasive pneumococcal disease in the elderly (0.30 / 1,000 for those over 65 years). These rates are projected to continue to increase in both the US and Europe (CDC 2013; Lamagni 2013).

[0005] One approach to prevent GBS disease among infants and the elderly is the use of polysaccharide-based vaccines. The implementation of maternal GBS prophylactic vaccination has the potential to prevent GBS disease among infants in the United States, regardless of whether IAP is used. Although polysaccharides can be immunogenic themselves, conjugation of polysaccharides with protein carriers has been used to improve immunogenicity, especially in infants and the elderly. Polysaccharide-protein conjugate vaccines are made using polysaccharides, generally from bacterial outer shells, that are associated with a protein carrier. The chemical bond of polysaccharides and protein carriers induces an immune response against bacteria that display the polysaccharides contained within the vaccine on their surface, thus preventing disease. Thus, vaccination using polysaccharides from pathogenic bacteria is a potential strategy to boost host immunity.

[0006] The polysaccharide coating of bacteria can vary widely even within a single bacterial species. For example, GBS has 10 different serotypes due to variations in the bacterial polysaccharide capsule. It is therefore desirable for a polysaccharide-based vaccine to consist of a panel of polysaccharides to ensure broad coverage of the different circulating strains.

[0007] The carrier protein may be a relevant protein antigen from the target pathogen that mounts a specific immune response against that pathogen, or it may be a generally immunogenic protein that further serves as an adjuvant or systemic immune response stimulator.

[0008] Individual monovalent polysaccharide-protein conjugates of GBS serotypes Ia, Ib, II, III, and V have been evaluated in phase I and II clinical trials in non-pregnant adults (Brigtsen, A. K. et al., Journal of Infectious Diseases, 2011, 144:131-132). es, 185(9):1277-1284(2002); Baker, CJ et al., J. Infect. Dis., 188(1):66-73(2003); Baker, CJ et al., J. Infect. Dis., 189(6):1103-1112(2004); Baker, CJ et al., Vaccine, 25(1):55-63(2007)). Bivalent II-TT and III-TT glycoconjugate vaccines, as well as trivalent vaccines including Ia-CRM197, Ib-CRM197 and III-CRM197 glycoconjugates, have also been investigated (Baker JID 2003; Clicaltrials.gov NCT01193920, NCT01412801 and NCT01446289). However, no GBS vaccine has yet been approved.

[0009] Moreover, the trivalent vaccine covers over 90% of invasive strains causing neonatal disease in South Africa (Madzivhandila, M. et al., PloS One, 6(3):e17861 (2011)), whereas these same serotypes represent only 62% and 66% of invasive isolates in North America and Europe, respectively, based on recent surveillance of neonatal isolates from a global collection of 901 samples collected between 2004 and 2013 from the Tigecycline Evaluation and Surveillance Trial (TEST, http: / / www.testsurveillance.com / ).

[0010] Analysis of strains obtained from TEST samples showed that 95% of the collected strains belonged to one of the five documented major serotypes (Ia, Ib, II, III, and V), with a further 3% being serotype IV. A series of publications also confirmed the emergence of serotype IV in the United States and Europe over the past decade (Diedrick, MJ et al., J. Clin. Microbiol., 48(9):3100-3104 (2010); Teatero (2014); Meehan, M. et al., European Journal of Clinical Microbiology & Infectious Diseases, 33(7):1155-1162 (2014); Florindo, C. et al., Euro Surveillance: Bulletin European sur les Maladies Transmissibles (European Communicable Disease Bulletin), 19(23) (2014); Palmiero, JK et al., Journal of Clinical Microbiology, 48(12):4397-4403 (2010)). A study investigating rectal / vaginal carriage in adults, a risk factor for transmission of GBS to infants, found that 97% of isolates belonged to one of these six serotypes, with serotype IV representing a frequency of approximately 4%. The study was designed to monitor carriage of beta-hemolytic streptococci (which encompass GBS), Clostridium difficile, and Staphylococcus aureus in healthy US adults (see Matson, MA, et al., ICAAC, Abstract I-306 (Washington, DC, Sep. 5-9, 2014)).

[0011] Similarly, analysis of TEST samples showed that 98% of US blood isolates from individuals aged 65 years or older belonged to the same six predominant serogroups. The most striking difference between elderly isolates and other populations is the serogroup distribution. For isolates from elderly patients, serogroup V strains comprise the largest group (34% vs. 18% for neonates or 18% for adult carrier strains).

[0012] Other studies have found that there is geographic dispersion of serotype prevalence. For example, serotype VI and VIII isolates were shown to be the predominant invasive species in healthy pregnant women in Japan (Lachenauer, CS et al., JID 179(4):1030-1033 (1999)). Summary of the Invention [Problem to be solved by the invention]

[0013] Thus, there is a need for polysaccharide-protein conjugate vaccines or monoclonal antibodies to confer passive immunity as a means to prevent or treat GBS disease, including that caused by the newly emerged serotype IV, among broad populations worldwide. [Means for solving the problem]

[0014] The present invention relates to novel immunogenic GBS polysaccharide-protein conjugates, methods for producing the conjugates, and immunogenic compositions comprising the conjugates, including the inventions disclosed in U.S. Provisional Patent Application No. 62 / 156,500, filed May 4, 2015, U.S. Provisional Patent Application No. 62 / 237,813, filed May 4, 2015, and U.S. Provisional Patent Application No. 62 / 237,820, filed October 6, 2015, which are incorporated by reference herein in their entireties. The following clauses describe certain aspects and embodiments of the present invention.

[0015] In one aspect, the present invention relates to an immunogenic polysaccharide-protein conjugate comprising a capsular polysaccharide from Group B Streptococcus (GBS) and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%. In another embodiment, the capsular polysaccharide may be up to about 40% desialylated (a sialylation level of greater than about 60%). In another embodiment, the capsular polysaccharide is selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX.

[0016] In additional embodiments, the immunogenic polysaccharide-protein conjugate comprises a capsular polysaccharide having about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 mM sialic acid per mM polysaccharide.

[0017] In another embodiment of the invention, the immunogenic conjugate comprises a capsular polysaccharide having a molecular weight between about 5 kDa and about 1,000 kDa, between about 25 kDa and about 750 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 200 kDa, or between about 100 kDa and about 400 kDa.

[0018] In further embodiments, the immunogenic conjugates of the invention have a molecular weight of between about 300 kDa and about 20,000 kDa, such as between about 1,000 kDa and about 15,000 kDa or between about 1,000 kDa and about 10,000 kDa.

[0019] In one embodiment, the immunogenic conjugate comprises a capsular polysaccharide having between about 0% and about 40% O-acetylation, such as less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% O-acetylation.

[0020] In one embodiment, the immunogenic conjugate comprises a capsular polysaccharide having at least about 0.1, 0.2, 0.3, 0.35 or about 0.4 mM O-acetate per mM saccharide repeat unit, hi another embodiment, the immunogenic conjugate comprises a capsular polysaccharide having less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM saccharide repeat unit.

[0021] In one embodiment, the immunogenic conjugate comprises CRM197 or tetanus toxoid as a carrier protein. In a specific embodiment, the carrier protein is CRM1 The number is 97.

[0022] An additional aspect of the invention is a method for isolating capsular polysaccharide comprising reacting an organic reagent with a cell broth containing a capsular polysaccharide producing bacteria. In one embodiment, the method further comprises a centrifugation step. In another embodiment, the method further comprises a filtration step. In certain embodiments, the capsular polysaccharide producing bacteria is selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis. In one embodiment, the hydroxylamine is selected from the group consisting of the amines listed in Table 2. In additional embodiments, the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine, diethylhydroxylamine, hydroxylamine, ethylenediamine, triethylenetetramine, 1,1,4,7,10,10 hexamethyltriethylenetetramine, and 2,6,10, trimethyl-2,6,10 triazaundecane. In yet another embodiment, the concentration of hydroxylamine is from about 5 mM to about 200 mM. In a further embodiment, the pH of the reaction is from about 5.5 to about 9.5. In an additional embodiment, the reaction occurs at a temperature of from about 20° C. to about 85° C. In another embodiment, the reaction reaction time is from about 10 hours to about 90 hours.

[0023] In one aspect, the invention relates to an immunogenic composition comprising a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharide from Group B Streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In another embodiment, the conjugate comprises GBS serotype IV and at least two additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In another embodiment, the conjugate comprises GBS serotype IV and at least three additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In another embodiment, the conjugate comprises GBS serotype IV and at least four additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In certain embodiments, the conjugate comprises capsular polysaccharides from serotypes Ia, Ib, II, III and IV. In further embodiments, the composition comprises GBS serotype V. In certain embodiments, the conjugate comprises capsular polysaccharides from serotypes Ia, Ib, II, III and V. In yet another embodiment, the immunogenic composition comprises six polysaccharide-protein conjugates, the conjugates comprising capsular polysaccharides from group B Streptococcus serotypes Ia, Ib, II, III, IV and V. One aspect of the invention relates to immunogenic compositions that are free of immune interference.

[0024] In one embodiment, the immunogenic composition further comprises a pharma- ceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic surfactant, inhibitor of free radical oxidation, carrier, or mixture thereof. In an additional embodiment, it comprises a buffer. The buffer may be HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine, or succinate. In a preferred embodiment, the buffer is histidine.

[0025] In another embodiment, the immunogenic composition further comprises a surfactant. The surfactant may be a polyoxyethylene sorbitan fatty acid ester, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20, or a polyoxyethylene alkyl ether. In a preferred embodiment, the surfactant is polysorbate-80.

[0026] In a further embodiment, the immunogenic composition further comprises an excipient. The excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol. In a preferred embodiment, the excipient is sodium chloride.

[0027] In yet another embodiment, the immunogenic composition further comprises an adjuvant. In one such embodiment, the adjuvant is an aluminum-based adjuvant or QS-21. In a preferred embodiment, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyl phosphate, and aluminum hydroxide. In a more preferred embodiment, the adjuvant is aluminum phosphate.

[0028] In one aspect of the invention, the immunogenic composition comprises a buffer, a surfactant, an excipient, and optionally an adjuvant, and the composition is buffered to a pH of about 6.0 to about 7.0. In another aspect, the immunogenic composition comprises histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, and the composition is buffered to a pH of about 6.0 to about 7.0. In a preferred embodiment, the immunogenic composition comprises about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride (NaCl), and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate. In a further aspect of the invention, the immunogenic composition comprises a dose of about 5 mcg / ml to about 50 mcg / ml.

[0029] In another aspect of the invention, the immunogenic composition is lyophilized, optionally in the presence of at least one excipient. In one embodiment, the at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol. In a preferred embodiment, the at least one excipient is selected from the group consisting of sucrose, mannitol and glycine. In a particular embodiment, the at least one excipient is sucrose. In one aspect, the lyophilized composition comprises about 1% (w / v) to about 10% (w / v), preferably more than about 5.5% (w / v), of the at least one excipient. In another embodiment, the lyophilized composition comprises an additional excipient. In one such embodiment, the additional excipient is mannitol or glycine. In a preferred embodiment, the lyophilized composition comprises about 1% (w / v) to about 10% (w / v) of the additional excipient. In yet another embodiment, the lyophilized composition is reconstituted with water, water for injection (WFI), an adjuvant suspension, or saline. In a particular embodiment, the diluent is a suspension of any of the adjuvants described herein, such as an aluminum-based adjuvant suspension, preferably an aluminum phosphate suspension.

[0030] Another aspect of the present invention is a method of eliciting an immune response against GBS, comprising administering to a subject an effective amount of an immunogenic composition as described herein. In one embodiment, the present invention is a method of preventing or reducing a disease or condition associated with GBS in a subject, comprising administering to a subject an effective amount of an immunogenic composition as described herein. In a particular embodiment, the subject is a woman planning to become pregnant or a woman who is pregnant. In one such embodiment, the pregnant woman is in the second half of pregnancy, such as at least 20 weeks or at least 27 weeks of pregnancy. In a preferred embodiment, the pregnant woman is between 27 and 36 weeks of pregnancy. In another embodiment, the subject is elderly, such as adults aged 50 years or older, 65 years or older, and 85 years or older. In a further embodiment, the subject is immunocompromised. In one aspect, the subject may have a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease. In a preferred embodiment, the group B streptococcus is Streptococcus agalactiae.

[0031] An additional aspect of the invention relates to antibodies that bind to capsular polysaccharides in the immunogenic compositions of the invention. In some embodiments, the antibodies are produced upon administration of the immunogenic composition to a subject. Another aspect relates to compositions comprising the antibodies of the invention.

[0032] A further aspect of the invention relates to a method of conferring passive immunity to a subject, the method comprising the steps of: (a) producing an antibody preparation using the immunogenic composition described herein; and (b) administering the antibody preparation to a subject to confer passive immunity.

[0033] One aspect of the present invention relates to a method of making the immunogenic polysaccharide-protein conjugate of the present invention, comprising the steps of (a) reacting a GBS capsular polysaccharide with an oxidizing agent to provide an activated polysaccharide, and (b) reacting the activated polysaccharide with a carrier protein to provide a polysaccharide-protein conjugate, wherein step (b) is carried out in a polar aprotic solvent. The solvent may be dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF) and hexamethylphosphoramide (HMPA). In a preferred embodiment, the solvent is dimethylsulfoxide (DMSO).

[0034] In one embodiment, the polysaccharide is reacted with 0.01 to 10.0 molar equivalents of an oxidizing agent. In a particular embodiment, the oxidizing agent is a periodate. In one such embodiment, the periodate is sodium periodate.

[0035] In another embodiment, the oxidation reaction is performed for between 1 hour and 50 hours. In a further embodiment, the temperature of the oxidation reaction is maintained between about 2° C. and about 25° C. In yet another embodiment, the oxidation reaction is performed in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) and Bis-Tris. In one such embodiment, the buffer has a concentration between about 1 mM and about 500 mM. In a particular embodiment, the oxidation reaction is performed at a pH between about 4.0 and about 8.0.

[0036] In a further aspect of the invention, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). In one such embodiment, N-chlorosuccinimide (NCS) is the co-oxidizing agent.

[0037] In one embodiment, step (a) of producing the immunogenic polysaccharide-protein conjugate of the invention further comprises the step of quenching the oxidation reaction by addition of a quenching agent. Included.

[0038] In another embodiment, the concentration of the polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL.

[0039] In an additional embodiment, the degree of oxidation (DO) of the activated polysaccharide is between 5 and 25.

[0040] In another aspect of the invention, the method further comprises the step of lyophilizing the activated polysaccharide. In one embodiment, the activated polysaccharide is lyophilized in the presence of a sugar selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit.

[0041] In a further aspect of the invention, step (b) of preparing the immunogenic polysaccharide-protein conjugate of the invention comprises the steps of (1) combining an activated polysaccharide with a carrier protein and (2) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate. In one embodiment, the concentration of the activated polysaccharide in step (2) is between about 0.1 mg / mL and about 10.0 mg / mL. In an additional embodiment, the initial ratio (wt / wt) of activated polysaccharide to carrier protein is between 5:1 and 0.1:1. In another embodiment, the reducing agent is selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride, sodium borohydride or zinc in the presence of a Bronsted or Lewis acid, an amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB), etc. In a preferred embodiment, the reducing agent is (NaBH3CN). In yet another embodiment, the amount of reducing agent is between about 0.1 and about 10.0 molar equivalents. In a further embodiment, the duration of the reduction reaction in step (2) is between 1 hour and 60 hours. In another embodiment, the temperature of the reduction reaction is maintained between 10°C and 40°C.

[0042] In an additional aspect of the invention, the method of making an immunogenic polysaccharide-protein conjugate further comprises a step of capping unreacted aldehydes by addition of borohydride (step (c)). In one embodiment, the amount of borohydride is between about 0.1 and about 10.0 molar equivalents. In another embodiment, the borohydride is selected from the group consisting of sodium borohydride (NaBH4), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride and magnesium borohydride. In a preferred embodiment, the borohydride is sodium borohydride (NaBH4). In a further embodiment, the duration of the capping step is between 0.1 and 10 hours. In yet another embodiment, the temperature of the capping step is maintained between about 15°C and about 45°C.

[0043] In another aspect of the present invention, the method further comprises purifying the polysaccharide-protein conjugate. In one embodiment, the polysaccharide-protein conjugate comprises less than about 40% free polysaccharide compared to the total amount of polysaccharide. In another embodiment, the ratio of polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.0. In a further embodiment, the degree of conjugation of the conjugate is between 2 and 15.

[0044] Another embodiment of the invention is a method of making an immunogenic polysaccharide-protein conjugate of the invention, comprising the steps of: (a) reacting an isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction of step (a) by the addition of a quenching agent to provide activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; and (e) capping any unreacted aldehydes by the addition of sodium borohydride (NaBH4), wherein steps (c), (d) and (e) are performed in DMSO. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 shows a comparison of the opsonic activity of sera and isolated IgG from mice immunized with GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197 monovalent vaccines. [Diagram 2] FIG. 1 shows the stability of GBS Ia-CRM197 (as indicated by % change in molecular weight using SEC MALLS) after accelerated storage (4 weeks) at 50° C. [Diagram 3] FIG. 1 shows the stability of GBS Ib-CRM197 (as indicated by % change in molecular weight using SEC MALLS) after accelerated storage (4 weeks) at 50° C. [Figure 4] FIG. 1 shows the stability of GBS II-CRM197 (as indicated by % change in molecular weight using SEC MALLS) after accelerated storage (4 weeks) at 50° C. [Diagram 5] FIG. 1 shows the stability of GBS III-CRM197 (as indicated by % change in molecular weight using SEC MALLS) after accelerated storage (4 weeks) at 50° C. [Figure 6]FIG. 1 shows the stability of GBS IV-CRM197 (as indicated by % change in molecular weight using SEC MALLS) after accelerated storage (4 weeks) at 50° C. [Figure 7] FIG. 1 shows the stability of GBS V-CRM197 (as indicated by % change in molecular weight using SEC MALLS) after accelerated storage (4 weeks) at 50° C. [Figure 8] FIG. 1 shows the stability (as indicated by free sialic acid) of GBS Ia-CRM197, GBS Ib-CRM197, GBS III-CRM197 and GBS IV-CRM197 after storage at 37° C. [Figure 9] FIG. 1 shows pH stability in hexavalent GBS vaccines using succinate as a buffer (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197). [Figure 10] FIG. 1 shows pH stability in hexavalent GBS vaccines using histidine as a buffer (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197). [Figure 11] FIG. 1 shows the effect of histidine buffer concentration in hexavalent GBS vaccines (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on aluminum binding of GBS conjugates at a dose of 10 mcg / ml. [Figure 12] FIG. 1 shows the effect of histidine buffer concentration in hexavalent GBS vaccines (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on aluminum binding of GBS conjugates at a dose of 40 mcg / ml. [Figure 13] FIG. 1 shows the effect of polysorbate-80 concentration in hexavalent GBS vaccines (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on the percent loss of total antigenicity upon agitation stress. [Figure 14] FIG. 1 shows the effect of aluminum concentration in the hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on GBS conjugate binding to aluminum. [Figure 15] FIG. 1 shows the effect of 5.5% (w / v) sucrose in a 10 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 16] FIG. 1 shows the effect of 7.0% (w / v) sucrose in a 10 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 17] FIG. 1 shows the effect of 8.5% (w / v) sucrose in a 10 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 18]FIG. 1 shows the effect of 5.5% (w / v) sucrose in a 50 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 19] FIG. 1 shows the effect of 7.0% (w / v) sucrose in a 50 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 20] FIG. 1 shows the effect of 8.5% (w / v) sucrose in a 50 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 21] FIG. 1 shows the effect of 7.0% (w / v) sucrose in a 40 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 22] FIG. 1 shows the effect of 2.0% (w / v) sucrose and 4.0% (w / v) mannitol in a 40 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 23]FIG. 1 shows the effect of 3.0% (w / v) sucrose and 3.0% (w / v) mannitol in a 40 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Figure 24] FIG. 1 shows the effect of 2.0% (w / v) sucrose and 4.0% (w / v) glycine in a 40 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. [Diagram 25] FIG. 1 shows the effect of 3.0% (w / v) sucrose and 3.0% (w / v) glycine in a 40 mcg / ml dose of lyophilized hexavalent GBS vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197) on antigenic recovery for each serotype. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] It is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0047] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

[0048] The terms used herein have meanings that are recognized and known to those of ordinary skill in the art; however, for convenience and completeness, certain terms and their meanings are explained below and throughout the specification.

[0049] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps described herein and / or that would become apparent to those skilled in the art upon reading this disclosure and so forth.

[0050] The term "about" or "approximately" means within a statistically meaningful range of values. Such a range may be within an order of magnitude, typically within 20%, and more typically within 10%, and even more typically within 5% of a given value or range. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily appreciated by one of ordinary skill in the art. Whenever a range is described within this application, all integers within the range are also contemplated as embodiments of the invention.

[0051] It should be noted that in this disclosure, terms such as "comprise," "include," "including," "containing," "containing" and the like may have the meaning ascribed to them in U.S. Patent Law, e.g., "includes," "included," "comprising," etc. Such terms refer to the inclusion of a particular ingredient or set of ingredients without the exclusion of any other elements. Terms such as "consisting essentially of" and "consisting essentially of" are used in the U.S. Patent Law. The terms "consisting of" and "consisting of" have the meanings ascribed to them in U.S. patent law, i.e., they are closed-ended. Thus, they refer to the inclusion of a particular ingredient or set of ingredients and the exclusion of all other ingredients.

[0052] The term "antigen" generally refers to an immunogenic substance capable of stimulating the production of an antibody or a T-cell response or both in an animal, including a biological molecule, usually a protein, peptide, polysaccharide, lipid or conjugate, that contains at least one epitope to which a cognate antibody can selectively bind, or in some cases a composition that is injected or absorbed into an animal. The immune response may be generated against the whole molecule or against one or more various portions of the molecule (e.g., epitopes or haptens). The term may be used to refer to individual molecules or homogeneous or heterogeneous populations of antigenic molecules. Antigens are recognized by antibodies, T-cell receptors, or other elements of specific humoral and / or cellular immunity. The term "antigen" encompasses all related antigenic epitopes. Epitopes of a given antigen can be identified using any number of epitope mapping techniques well known to those of skill in the art (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, ed., 1996) Humana Press, Totowa, NJ). For example, linear epitopes can be determined, e.g., by simultaneously synthesizing a large number of peptides on a solid support, peptides corresponding to portions of a protein molecule, and reacting the peptides with an antibody while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen, HM et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1984); Geysen, HM et al., Molec. Immunol., 23(7):709-715 (1986), all of which are incorporated herein by reference in their entireties. Similarly, conformational epitopes can be identified by determining the spatial conformation of amino acids, such as by, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols, supra).Furthermore, for purposes of the present invention, "antigen" may also be used to refer to proteins that include modifications (generally conservative in nature, but which may be non-conservative) to the native sequence, such as deletions, additions, and substitutions, so long as the protein maintains its ability to elicit an immunological response. These modifications may be deliberate, such as through site-directed mutagenesis, or through specific synthetic procedures, or through genetic engineering approaches, or may be accidental, such as through mutations of the host that produces the antigen. Furthermore, antigens may be derived, obtained, or isolated from microorganisms, such as bacteria, or may be whole organisms. Similarly, oligonucleotides or polynucleotides expressing antigens, such as in nucleic acid immunization applications, are also included in the definition. Synthetic antigens, such as polyepitopes, contiguous epitopes, and other recombinant or synthetically derived antigens, are also encompassed (Bergmann, C. et al., Eur. J. Immunol., 23(11):2777-2781 (1993); Bergmann, CC et al., J. Immunol., 157(8):3242-3249 (1996); Suhrbier, A., Immunol. and Cell Biol., 75(4):402-408 (1997)).

[0053] The terms "vaccine" or "vaccine composition" are used interchangeably and refer to a pharmaceutical composition that includes at least one immunogenic composition that elicits an immune response in an animal.

[0054] capsular polysaccharide As used herein, the term "sugar" refers to a single sugar moiety or unit, as well as combinations of two or more single sugar moieties or units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. The term "sugar" may be used interchangeably with the term "carbohydrate." Polysaccharides may be linear or branched.

[0055] "Monosaccharide" as used herein refers to a single sugar residue in an oligosaccharide. The term "disaccharide" as used herein refers to a polysaccharide composed of two monosaccharide units or moieties linked together by a glycosidic bond.

[0056] In one embodiment, the polysaccharide is an oligosaccharide (OS). "Oligosaccharide" as used herein refers to a compound containing two or more monosaccharide units or moieties. Within the context of an oligosaccharide, each monomer unit or moiety is a monosaccharide that is or can be linked to another monosaccharide unit or moiety via a hydroxyl group. Oligosaccharides can be prepared either by chemical synthesis from protected single residue sugars or by chemical degradation of biologically produced polysaccharides. Alternatively, oligosaccharides can be prepared by in vitro enzymatic methods.

[0057] In a preferred embodiment, the polysaccharide is a polysaccharide (PS), which refers to a linear or branched polymer of at least five monosaccharide units or moieties. For clarity, larger numbers of repeating units (where n is greater than about 5, such as greater than about 10) are referred to herein as polysaccharides.

[0058] In one embodiment, the polysaccharide is a cell surface polysaccharide. Cell surface polysaccharide refers to a polysaccharide having at least a portion located on the outermost bacterial cell membrane or bacterial cell surface, including the peptidoglycan layer, cell wall and capsule. Typically, cell surface polysaccharides are associated with eliciting an immune response in vivo. Cell surface polysaccharides may be "cell wall polysaccharides" or "capsular polysaccharides". Cell wall polysaccharides typically form a discontinuous layer on the bacterial surface.

[0059] In one embodiment, the polysaccharide is a capsular polysaccharide. Capsular polysaccharides are glycopolymers that include repeating units of one or more monosaccharides joined by glycosidic bonds. Capsular polysaccharides typically form a capsule-like layer around the bacterial cell. "Capsular polysaccharide" or "capsule polysaccharide" refers to the polysaccharide capsule that is on the exterior of the cell wall of most isolates of streptococci. For example, all GBS capsular polysaccharides have a branched repeating structure with terminal α2-3-linked sialic acid that is required for bacterial virulence. Capsular-associated sialic acid (quantified by HPLC assay) was detected in more than 94% of invasive neonatal isolates from TEST cultured in vitro.

[0060] The inventors have discovered that the sialic acid level of GBS capsular polysaccharide is an important feature for generating an immune response. Prior disclosures have provided conflicting information regarding the sialic acid level for serotype V, only the observation that desialylated serotype V is preferred (International Patent Application Publication No. WO2012 / 035519), and that a sialic acid content of more than 50% for serotype V can be used (International Patent Application Publication No. WO2014 / 053612). However, none of these references describes the importance of the sialic acid level for at least the majority of the GBS polysaccharide to immunogenicity. The inventors have surprisingly found that GBS capsular polysaccharide generates an immune response ( We found that about 60% or more sialic acid was required prior to conjugation to provide a sialic acid content of 100% or greater than about 95%. Even a sialic acid level of 58%, which is within the previously disclosed range for serotype V, adversely affected immunogenicity.

[0061] Thus, in one embodiment of the invention, the capsular polysaccharides comprise their native sialic acid levels of about 100% or greater than about 95%, etc. In another embodiment, the capsular polysaccharides may be desialylated, such as up to about 40% (greater than about 60% sialylation level), for example up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), and up to about 5% (greater than about 95% sialylation level).

[0062] It should be noted that a sialic acid level of 100% corresponds to about 1.0 mM sialic acid per mM polysaccharide. Thus, a capsular polysaccharide may have about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide. In further embodiments, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, e.g., at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0063] The terminal sialic residues of some capsular polysaccharide (CP) serotypes are partially O-acetylated (OAc) (Lewis, AL et al., Proceedings of the National Academy of Sciences USA, 101(30):11123-8 (2004)). Serotypes Ib, III, IV, V, VI and IX are partially O-acetylated (up to about 40%), whereas serotypes Ia, II and VII have little or no O-acetylation (less than about 5%) (Lewis 2004). In one embodiment of the invention, the capsular polysaccharides comprise their natural O-acetylation level (from about 0% to about 40%). In another embodiment, the capsular polysaccharides may be O-deacetylated (less than about 5%). The degree of O-acetylation of polysaccharides or oligosaccharides can be determined by any method known in the art, for example, by proton NMR (Lemercinier, X. et al., Carbohydrate Research, 296:83-96 (1996); Jones, C. et al., Journal of Pharmaceutical and Biomedical Analysis, 30:1233-1247 (2002); International Patent Application Publication Nos. WO2005 / 033148 and WO00 / 56357). Another commonly used method is described by Hestrin, S., J. Biol. Chem., 180:249-261 (1949).

[0064] It should also be noted that 100% O-acetate corresponds to about 1.0 mM O-acetate per mM saccharide repeat unit. Thus, a partially O-acetylated polysaccharide contains at least about 0.1, 0.2, 0.3, 0.35 or about 0.4 mM O-acetate per mM saccharide repeat unit. A polysaccharide that is O-deacetylated contains less than about 0.01, 0.02, 0.03, 0.04 or 0.05 mM O-acetate per mM saccharide repeat unit.

[0065] Streptococcal microorganisms capable of causing invasive disease are also generally capable of producing CPs that encapsulate the bacteria and enhance their resistance to clearance by the host's innate immune system. CPs encapsulate bacterial cells within a protective capsule that renders the bacteria resistant to phagocytosis and intracellular killing. Capsular polysaccharides serve to encase the vacuole. Bacteria lacking a capsule are more susceptible to phagocytosis. Capsular polysaccharides are frequently important virulence factors for many bacterial pathogens, including Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Staphylococcus aureus.

[0066] Capsular polysaccharides can be used to serotype specific species of bacteria. Typing to specific structures or unique epitope features of the capsular polysaccharide is usually accomplished by reaction with specific antisera or monoclonal antibodies produced. There are 10 GBS serotypes: Ia, Ib, and II-IX (Ferrieri, P. et al., Emerg. Infect. Dis. [Internet], 19(4) (2013), available at http: / / wwwnc.cdc.gov / eid / article / 19 / 4 / 12-1572_article).

[0067] In one embodiment of the invention, the polysaccharide is isolated from Streptococcus agalactiae. The polysaccharide may be isolated from any encapsulated strain of S. agalactiae, such as 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524, MB4052 (ATCC Accession No. 31574), H36B (ATCC Accession No. 12401), S40, S42, MB4053 (ATCC Accession No. 31575), M709, 133, 709, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1111, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1310, 1320, 1330, 1400, 1410 357, PFEGBST0267, MB4055 (ATCC Accession No. 31576), 18RS21 (ATCC Accession No. BAA-1175), S16, S20, V8 (ATCC Accession No. 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB4082 (ATCC Accession No. 31577), M132, 110, M781 (ATCC Accession No. BAA-22), D136C(3)(ATCC Accession No. C accession no. 12403), M782, S23, 120, MB4316 (M-732; ATCC accession no. 31475), M132, K79, COH1 (ATCC accession no. BAA-1176), PFEGBST0563, 3139 (ATCC accession no. 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession no. BAA-23), CJB112, 2603V / R (ATCC Accession No. BAA-611), NCTC10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B4589, B4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62 and IT-PW-64, etc.

[0068] The polysaccharides described herein may be isolated by methods known in the art, including, for example, the methods described herein. As used herein, "isolated" refers to being obtained and separated from a particular source. The term "isolated" further refers to being free from its respective naturally occurring form, situation and / or environment. For example, "isolated from streptococci" refers to a material obtained and separated from streptococcal cells. An isolated polysaccharide is not naturally occurring. The term "isolated" means that the material has been removed from its original environment (e.g., from the natural environment if it is naturally occurring, or from its host organism if it is a recombinant entity, or transported from one environment to a different environment). For example, an "isolated" capsular polysaccharide, protein or peptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals if chemically synthesized or present in a mixture as part of a chemical reaction. In the present invention, a protein or polysaccharide is a protein or peptide that is substantially free of immunogenicity, immunogenicity, or immunogenicity. It may be isolated from bacterial cells or from cell debris so as to be provided in a form useful in the manufacture of compositions. The term "isolated" or "isolating" may include purifying or purification, including methods for purifying isolated polysaccharides known in the art and / or described herein. The term "substantially free of cellular material" includes preparations of a polypeptide / protein in which the polypeptide / protein is separated from cellular components of the cells from which it was isolated or recombinantly produced. Thus, a capsular polysaccharide, protein or peptide that is substantially free of cellular material includes preparations of capsular polysaccharides, proteins or peptides having less than about 30%, 20%, 10%, 5%, 2.5% or 1% (by dry weight) of contaminating proteins or polysaccharides or other cellular material. If the polypeptide / protein is recombinantly produced, it is preferably also substantially free of culture medium, i.e., culture medium represents less than about 20%, 10% or 5% of the volume of the protein preparation. When a polypeptide / protein or polysaccharide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein or polysaccharide. Thus, such preparations of a polypeptide / protein or polysaccharide have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide / protein or polysaccharide fragment of interest.

[0069] In one embodiment of the invention, the polysaccharide is isolated from bacteria. In another embodiment of the invention, the polysaccharide is recombinantly produced. In a further embodiment, the polysaccharide is synthetic or chemically synthesized according to conventional methods. In yet another embodiment of the invention, the polysaccharide is prepared by cloning and expressing a biosynthetic pathway for producing the polysaccharide followed by expression in a surrogate host. In one embodiment, the polysaccharide is immunogenic. For example, the inventors have discovered that each of the polysaccharides described herein is capable of eliciting or eliciting an immune response. The term "immunogenic" refers to the ability to initiate, trigger, cause, enhance, improve, and / or enhance a humoral and / or cell-mediated immune response in a mammal. In one embodiment, the mammal is a human, a primate, a rabbit, a pig, a mouse, etc.

[0070] The molecular weight of the capsular polysaccharide is a consideration for use in immunogenic compositions. High molecular weight capsular polysaccharides can elicit certain antibody immune responses due to the higher valency epitopes present on the antigen surface. Isolation and purification of high molecular weight capsular polysaccharides are contemplated for use in the conjugates, compositions and methods of the invention.

[0071] However, in one embodiment, the polysaccharide may be sized to a lower molecular weight (MW) range than the molecular weight of the native capsular polysaccharide prior to conjugation with the carrier protein. The size of the purified capsular polysaccharide is reduced to produce a conjugate with advantageous filtration properties and / or yield.

[0072] In one such embodiment, the size of the purified capsular polysaccharide is reduced by high pressure homogenization, which achieves high shear rates by pumping the process stream through a channel with sufficiently small dimensions. The shear rate is increased by using a large applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0073] In one embodiment, the polysaccharides described herein are capable of inducing opsonic activity. In another embodiment, the polysaccharides described herein are capable of inducing opsonic and phagocytic activity (e.g., opsonophagocytic activity).

[0074] Opsonic activity or opsonization is the process by which an opsonin (e.g., an antibody or complement factor) binds to a target cell. It refers to the process of binding to an antigen (e.g., an isolated polysaccharide described herein), which facilitates binding of the antigen to a phagocyte or phagocytic cell (e.g., macrophage, dendritic cell, and polymorphonuclear leukocyte (PMNL)). Some bacteria, such as encapsulated bacteria that are not typically phagocytosed due to the presence of a capsule, become more susceptible to recognition by phagocytes when coated with opsonic antibodies. In one embodiment, the polysaccharide elicits an opsonic immune response, such as antibodies. In one embodiment, the opsonic activity is against gram-positive cocci, preferably against a Streptococcus species, more preferably against at least one strain of S. agalactiae.

[0075] In yet another embodiment, the polysaccharides described herein are capable of eliciting a bactericidal immune response, hi one embodiment, the bactericidal activity is against gram-positive cocci, preferably against Streptococcus species, more preferably against at least one strain of S. agalactiae.

[0076] Methods for measuring opsonization, phagocytosis and / or bactericidal activity are known in the art, for example, by measuring reduction in bacterial load in vivo (e.g., by measuring bacteremia levels in mammals inoculated with Streptococcus species) and / or by measuring bacterial cell killing in vitro (e.g., in vitro opsonophagocytosis assays). In one embodiment, the polysaccharide is capable of eliciting opsonization, phagocytosis and / or bactericidal activity compared to an appropriate control, such as, for example, compared to antisera raised against heat-killed Gram-positive cocci.

[0077] Serotype Ia One embodiment encompasses serotype Ia GBS capsular polysaccharide. The structure of serotype Ia can be depicted as follows:

[0078] [ka]

[0079] The molecular weight of the serotype Ia capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa. between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about between about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 45 between about 0 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and about 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa.In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 100 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0080] In certain embodiments, a high pressure homogenization process is used to reduce the size of native GBS capsular polysaccharide serotype Ia while preserving structural features of the polysaccharide, such as sialic acid.

[0081] In one embodiment of the invention, the serotype Ia capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0082] In another embodiment, the serotype Ia capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide. In a further embodiment, the capsular polysaccharide has, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 ... It may have at least about 0.9 mM sialic acid per mM, or at least about 0.95 mM sialic acid per mM polysaccharide, and so forth.

[0083] Serotype Ia capsular polysaccharides are less than about 5% O-acetylated. Some exemplary strains of serotype Ia capsular polysaccharides of the invention include 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524 and MB4052 (ATCC Accession No. 31574).

[0084] Serotype Ib One embodiment encompasses serotype Ib GBS capsular polysaccharide. The structure of serotype Ib can be depicted as follows:

[0085] [ka]

[0086] The molecular weight of the serotype Ib capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 30 ... Da to about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, about 75 kDa Da to about 500 kDa, between about 75 kDa to about 450 kDa, between about 75 kDa to about 400 kDa, between about 75 kDa to about 350 kDa, between about 75 kDa to about 300 kDa, between about 75 kDa to about 250 kDa, between about 75 kDa to about 200 kDa, between about 100 kDa to about 750 kDa, between about 100 kDa to about 700 kDa, between about 100 kDa to about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 6 between 50 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and the like. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0087] In one embodiment of the invention, the serotype Ib capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0088] In another embodiment, the serotype Ib capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0089] Serotype Ib capsular polysaccharides are between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype Ib capsular polysaccharides of the invention include H36B (ATCC Accession No. 12401), S40, S42, MB4053 (ATCC Accession No. 31575), M709, 133, 7357 and PFEGBST0267.

[0090] Serotype II One embodiment encompasses serotype II GBS capsular polysaccharide. The structure of serotype II can be depicted as follows:

[0091] [ka]

[0092] The molecular weight of the serotype II capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of these is contemplated as an embodiment of the present disclosure.

[0093] In one embodiment of the invention, the serotype II capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0094] In another embodiment, the serotype II capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0095] Serotype II capsular polysaccharides are less than about 5% O-acetylated. Some exemplary strains of serotype II capsular polysaccharides of the invention include MB4055 (ATCC Accession No. 31576), 18RS21 (ATCC Accession No. BAA-1175), S16, S20, V8 (ATCC Accession No. 12973), DK21, DK23, UAB, 5401 and PFEGBST0708.

[0096] Serotype III One embodiment encompasses serotype III GBS capsular polysaccharide. The structure of serotype III can be depicted as follows:

[0097] [ka]

[0098] The molecular weight of the serotype III capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa. between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 45 between about 0 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and about 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa.In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 100 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0099] In certain embodiments, a high pressure homogenization process is used to reduce the size of native GBS capsular polysaccharide serotype III while preserving structural features of the polysaccharide, such as sialic acid.

[0100] In one embodiment of the invention, the serotype III capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0101] In another embodiment, the serotype III capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide. In a further embodiment, the capsular polysaccharide has, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 ... It may have at least about 0.9 mM sialic acid per mM saccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, and so forth.

[0102] Serotype III capsular polysaccharides are between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype III capsular polysaccharides of the invention include MB4082 (ATCC Accession No. 31577), M132, 110, M781 (ATCC Accession No. BAA-22), D136C(3) (ATCC Accession No. 12403), M782, S23, 120, MB4316 (M-732; ATCC Accession No. 31475), M132, K79, COH1 (ATCC Accession No. BAA-1176) and PFEGBST0563.

[0103] Serotype IV One embodiment encompasses serotype IV GBS capsular polysaccharide. The structure of serotype IV can be depicted as follows:

[0104] [ka]

[0105] The molecular weight of the serotype IV capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa, between about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about Between about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, Between 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and about 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0106] In one embodiment of the invention, the serotype IV capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0107] In another embodiment, the serotype IV capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0108] Serotype IV capsular polysaccharides are between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype IV capsular polysaccharides of the invention include 3139 (ATCC Accession No. 49446), CZ-NI-016, and PFEGBST0961.

[0109] Serotype V One embodiment encompasses serotype V GBS capsular polysaccharide. The structure of serotype V can be depicted as follows:

[0110]

change

[0111] The molecular weight of the serotype V capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 5 between about 0 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0112] In one embodiment of the invention, the serotype V capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0113] In another embodiment, the serotype V capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0114] Serotype V capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype V capsular polysaccharide of the invention include 1169-NT1, CJB111 (ATCC Accession No. BAA-23), CJB112, 2603V / R (ATCC Accession No. BAA-611), NCTC10 / 81, CJ11, and PFEGBST0837.

[0115] Serotype VI The GBS serotype VI capsular polysaccharide has been described by von Hunolstein, C. et al., Infection and Immunity, 61(4):1272-1280 (1993), the disclosure of which is incorporated herein by reference in its entirety. The structure of serotype VI can be depicted as follows:

[0116] [ka]

[0117] The molecular weight of the serotype VI capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about between about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, about 100 kDa between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa Da, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0118] In one embodiment of the invention, the serotype VI capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0119] In another embodiment, the serotype VI capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0120] Serotype VI capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype VI capsular polysaccharide of the invention include 118754, 114852, 114862, 114866, 118775, B4589, B4645, SS1214 and CZ-PW-119.

[0121] Serotype VII The GBS serotype VII capsular polysaccharide has been described by Kogan, G. et al., Carbohydrate Research, 277(1):1-9 (1995), the disclosure of which is incorporated herein by reference in its entirety. The repeating unit of serotype VII is as follows:

[0122] [ka]

[0123] The molecular weight of the serotype VII capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about Between about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 500 kDa, Da to about 400 kDa, between about 100 kDa to about 350 kDa, between about 100 kDa to about 300 kDa, between about 200 kDa to about 750 kDa, between about 200 kDa to about 700 kDa, between about 200 kDa to about 650 kDa, between about 200 kDa to about 600 kDa, between about 200 kDa to about 550 kDa, between about 200 kDa to about 500 kDa, between about 200 kDa to about 450 kDa, between about 200 kDa to about 400 kDa, between about 250 kDa to about 750 kDa, between about 250 kDa to about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0124] In one embodiment of the invention, the serotype VII capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0125] In another embodiment, the serotype VII capsular polysaccharide is, prior to conjugation, In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, prior to conjugation.

[0126] Serotype VII capsular polysaccharide is less than about 5% O-acetylated. Some exemplary strains of serotype VII capsular polysaccharide of the invention include 7271 and CZ-PW-045.

[0127] Serotype VIII The GBS serotype VIII capsular polysaccharide has been described by Kogan, G. et al., The Journal of Biological Chemistry, 271(15):8786-8790 (1996), the disclosure of which is incorporated herein by reference in its entirety. The repeating unit of serotype VIII is as follows:

[0128] [ka]

[0129] The molecular weight of the serotype VIII capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 50 ... between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 4 ... Da to about 400 kDa, between about 100 kDa to about 350 kDa, between about 100 kDa to about 300 kDa, between about 200 kDa to about 750 kDa, between about 200 kDa to about 700 kDa, between about 200 kDa to about 650 kDa, between about 200 kDa to about 600 kDa, between about 200 kDa to about 550 kDa, between about 200 kDa to about 500 kDa, between about 200 kDa to about 450 kDa, between about 200 kDa to about 400 kDa, between about 250 kDa to about 750 kDa, between about 250 kDa to about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa. Any integer within any of these is contemplated as an embodiment of the present disclosure.

[0130] In one embodiment of the invention, the serotype VIII capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0131] In another embodiment, the serotype VIII capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0132] The serotype VIII capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of the serotype VIII capsular polysaccharide of the present invention include JM9130013 and JM9130672.

[0133] Serotype IX The GBS serotype IX capsular polysaccharide has been described by Berti, F. et al., The Journal of Biological Chemistry, 289(34):23437-2348 (2014), the disclosure of which is incorporated herein by reference in its entirety. The structure of serotype IX can be depicted as follows:

[0134] [ka]

[0135] The molecular weight of the serotype IX capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 50 ... between about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, about 100 kDa between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa Da, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and about 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0136] In one embodiment of the invention, the serotype IX capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0137] In another embodiment, the serotype IX capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0138] Serotype IX capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype IX capsular polysaccharide of the invention include IT-NI-016, IT-PW-62 and IT-PW-64.

[0139] Polysaccharide-protein conjugates As used herein, a "conjugate" comprises a capsular polysaccharide, usually of a desired molecular weight range, and a carrier protein, where the capsular polysaccharide is conjugated to the carrier protein. The conjugate may or may not contain some amount of free capsular polysaccharide. As used herein, "free capsular polysaccharide" refers to the capsular polysaccharide that is non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped within or by) the capsular polysaccharide-carrier protein to which it is conjugated. The terms "free capsular polysaccharide," "free polysaccharide," and "free saccharide" may be used interchangeably and are intended to convey the same meaning. Regardless of the nature of the carrier molecule, the carrier molecule can be conjugated to the capsular polysaccharide, either directly or via a linker. As used herein, "conjugate," "conjugate" and "conjugate" are used interchangeably. "Covalently binding" and "conjugating" refer to the process by which bacterial capsular polysaccharides are covalently attached to a carrier molecule. Conjugation enhances the immunogenicity of bacterial capsular polysaccharides. Conjugation can be carried out according to the methods described below or by processes known in the art.

[0140] "Conjugate immunogenic composition" as used herein refers to an immunogenic composition in which the immunogenic material includes an antigenic polysaccharide covalently linked to a carrier protein to provide a polysaccharide-protein conjugate. In one embodiment, the polysaccharide-protein conjugates of the invention may be formulated as multivalent immunogenic compositions.

[0141] As used herein, the term "molecular weight" of a polysaccharide or of a carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0142] As used herein, "polysaccharide-protein conjugate" refers to a polysaccharide molecule conjugated to a protein carrier molecule through one or more covalent bonds. It may be desirable to conjugate the polysaccharide to a protein from another species known to be immunogenic in the target host. Thus, in one embodiment, the carrier molecule is a carrier protein. As defined herein, such a foreign protein is referred to as a "carrier protein". The carrier protein serves to enhance the antigenicity and immunogenicity of the polysaccharide. As used herein, the term "carrier effect" refers to the process in which the antigenicity and immunogenicity of a weakly immunogenic or non-immunogenic molecule is enhanced by being combined with a more immunogenic molecule (e.g., a heterologous protein) as a carrier. In this case, the polysaccharide in the combined polysaccharide-protein conjugate is more immunogenic than when presented alone. The carrier protein contains T cell epitopes to stimulate T cells that help generate an antibody response.

[0143] "Carrier protein" or "protein carrier" as used herein refers to any protein molecule that can be conjugated with an antigen (such as a capsular polysaccharide) against which an immune response is desired. Conjugation of an antigen, such as a polysaccharide, with a carrier protein can make the antigen immunogenic. The carrier protein is preferably a protein that is non-toxic and non-reactogenic and can be obtained in sufficient quantity and purity. Examples of carrier proteins are toxins, toxoids, or any mutant cross-reactive material (CRM197) of toxins from tetanus, diphtheria, whooping cough, Pseudomonas species, E. coli, Staphylococcus species, and Streptococcus species. The carrier protein must be suitable for standard conjugation procedures. In a specific embodiment of the present invention, CRM197 is used as the carrier protein.

[0144] Cross-reacting materials or CRMs are particularly useful in some embodiments of the present invention. Genetically modified proteins can be produced that are antigenically similar to certain bacterial toxins, but are non-toxic. These are referred to as "cross-reacting materials" or CRMs. CRM197 (Wyeth / Pfizer Inc., Sanford, NC) is notable because it has a single amino acid change from the native diphtheria toxin and is immunologically indistinguishable from it. See Pappenheimer, AM et al., Immunochem., 9(9):891-906 (1972); U.S. Patent No. 5,614,382, the disclosures of which are incorporated herein by reference in their entireties. CRM197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from a culture of Corynebacterium diphtheriae strain C7 (β197) grown in a casamino acid and yeast extract-based medium. CRM197 was isolated by ultrafiltration. The CRM197 protein is purified via ammonium sulfate precipitation and ion exchange chromatography. A culture of C. diphtheriae strain C7 (β197) which produces the CRM197 protein has been deposited at the American Type Culture Collection, Rockville, Maryland, and has been assigned the accession number ATCC 53281. Other diphtheria toxoids are also suitable for use as carrier proteins. CRM3201 is a genetically engineered variant of pertussis toxin. See Black, WJ et al., Science, 240(4852):656-659 (1988), the disclosures of which are incorporated herein by reference in their entirety.

[0145] In addition to diphtheria toxoid (DT), CRM197 and pertussis toxoid, further examples of carrier proteins include tetanus toxoid (TT), cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004 / 083251), E. coli heat labile toxoid (LT), E. coli heat stable toxoid (ST), pneumolysin from S. pneumoniae (wild type or a virulence-reduced mutant), pneumococcal surface protein A (PspA), pneumococcal adhesin protein A (PsaA), C5a peptidase from Streptococcus, hemolysin from Staphylococcus aureus, nontypeable Haemophilus influenzae (NTHi) protein, Haemophilus influenzae influenzae protein D, Clostridium perfringens exotoxin / toxoid, Hepatitis B surface antigen, Hepatitis B core antigen, Rotavirus VP7 protein, and respiratory syncytial virus F and G proteins, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derivative (PPD) of tuberculin, and Pseudomonas exotoxin or derivatives thereof, including recombinantly produced non-toxic mutant Pseudomonas aeruginosa exotoxin A. Bacterial outer membrane proteins, such as outer membrane protein complex c (OMPC), porins, transferrin-binding proteins, etc., or C. difficile enterotoxin (toxin A) and cytotoxin (toxin B) can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), can also be used as carrier proteins. In a preferred embodiment, the carrier protein is diphtheria toxoid. More preferably, the carrier protein is CRM197. In another embodiment of the present invention, the carrier protein is tetanus toxoid.

[0146] For the synthesis of a multivalent conjugate immunogenic composition, a polysaccharide-protein conjugate can be produced by conjugating a mixture of polysaccharides purified from two different species of bacteria with a carrier protein. Alternatively, a multivalent conjugate immunogenic composition can be produced by combining polysaccharides purified from two or more different serotypes of the same bacterium and conjugating them as a mixture with a carrier protein. Alternatively, polysaccharide-protein conjugates produced by reacting a single type of polysaccharide with a carrier protein in separate reactions using different polysaccharides can be mixed. Thus, a multivalent immunogenic composition can include a carrier protein carrying a homogeneous or heterogeneous population of bound polysaccharides.

[0147] After conjugation of the capsular polysaccharide with the carrier protein, the polysaccharide-protein conjugate is purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various techniques, including, for example, concentration / hemodiafiltration operations, precipitation / elution, column chromatography and depth filtration.

[0148] As mentioned above, the present invention relates to a conjugate comprising a GBS capsular polysaccharide conjugated to a carrier protein. One embodiment of the present invention provides a conjugate comprising a GBS serotype IV capsular polysaccharide conjugated to a carrier protein and at least one further conjugate comprising a GBS serotype Ia capsular polysaccharide conjugated to a carrier protein, a GBS serotype Ib capsular polysaccharide conjugated to a carrier protein, a GBS serotype II capsular polysaccharide conjugated to a carrier protein, a GBS serotype III capsular polysaccharide conjugated to a carrier protein, a GBS serotype V capsular polysaccharide conjugated to a carrier protein, a GBS serotype VI capsular polysaccharide conjugated to a carrier protein, a GBS serotype VII capsular polysaccharide conjugated to a carrier protein, a GBS serotype VIII capsular polysaccharide conjugated to a carrier protein, or a GBS serotype IX capsular polysaccharide conjugated to a carrier protein. In one aspect of the invention, the polysaccharide has a molecular weight between about 5 kDa and 1,000 kDa, the conjugate has a molecular weight between about 300 kDa and about 20,000 kDa, and the conjugate comprises less than about 40% free polysaccharide relative to the total polysaccharide, hi one embodiment, the conjugate comprises less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free polysaccharide relative to the total polysaccharide.

[0149] In one embodiment, the serotype Ia, Ib, II, III, IV, V, VI, VII, VIII and / or IX polysaccharides are, prior to conjugation, between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa. between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, about 100 kDa between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about and / or about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and about 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa.Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0150] In one embodiment, the conjugate has a molecular weight of between about 300 kDa and about 20,000 kDa, e.g., between about 300 kDa and about 15,000 kDa, between about 300 kDa and about 10,000 kDa, between about 300 kDa and about 9,000 kDa, between about 300 kDa and about 8,000 kDa, between about 300 kDa and about 7,000 kDa, between about 300 kDa and about 6,000 kDa, between about 300 kDa and about 5,000 kDa, between about 300 kDa and about 4,000 kDa, between about 300 kDa and about 3,000 kDa, between about 30 between about 0 kDa and about 2,000 kDa, between about 300 kDa and about 1,000 kDa, between about 500 kDa and about 20,000 kDa, between about 500 kDa and about 15,000 kDa, between about 500 kDa and about 10,000 kDa, between about 500 kDa and about 9,000 kDa, between about 500 kDa and about 8,000 kDa, between about 500 kDa and about 7,000 kDa, between about 500 kDa and about 6,000 kDa, between about 500 kDa and about 5,000 kDa, between about 500 kDa and about 4,000 kDa, between about 500 kDa and about 3,000 kDa between about 500 kDa and about 2,000 kDa, between about 500 kDa and about 1,000 kDa, between about 1,000 kDa and about 20,000 kDa, between about 1,000 kDa and about 15,000 kDa, between about 1,000 kDa and about 10,000 kDa, between about 1,000 kDa and about 9,000 kDa, between about 1,000 kDa and about 8,000 kDa, between about 1,000 kDa and about 7,000 kDa, between about 1,000 kDa and about 6,000 kDa, between about 1,000 kDa and about 5,000 kDa, between about 1,500 kDa and about 20,000 kDa kDa, between about 1,500 kDa and about 15,000 kDa, between about 1,500 kDa and about 10,000 kDa, between about 1,500 kDa and about 9,000 kDa, between about 1,500 kDa and about 8,000 kDa, between about 1,500 kDa and about 7,000 kDa, between about 1,500 kDa and about 6,000 kDa, between about 1,500 kDa and about 5,000 kDa, between about 2,000 kDa and about 20,000 kDa, between about 2,000 kDa and about 15,000 kDa, between about 2,000 kDa and about 10,000 kDa, about 2,000 kDa between about 2,000 kDa and about 8,000 kDa, between about 2,000 kDa and about 7,000 kDa, between about 2,000 kDa and about 6,000 kDa, between about 2,500 kDa and about 20,000 kDa, between about 2,500 kDa and about 15,000 kDa, between about 2,500 kDa and about 10,000 kDa, between about 2,500 kDa and about 9,000 kDa, between about 2,500 kDa and about 8,000 kDa, between about 2,500 kDa and about 7,000 kDa, between about 2,500 kDa and about 6,000 kDa, between about 3,The molecular weight may be between about 3,000 kDa and about 20,000 kDa, between about 3,000 kDa and about 15,000 kDa, between about 3,000 kDa and about 10,000 kDa, between about 3,000 kDa and about 9,000 kDa, between about 3,000 kDa and about 8,000 kDa, between about 3,000 kDa and about 7,000 kDa, or between about 3,000 kDa and about 6,000 kDa.

[0151] In certain embodiments, the GBS serotype IV capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0152] In certain embodiments, the GBS serotype Ia capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0153] In certain embodiments, the GBS serotype Ib capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0154] In certain embodiments, the GBS serotype II capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0155] In certain embodiments, the GBS serotype III capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0156] In certain embodiments, the GBS serotype V capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0157] In one embodiment, the GBS serotype VI capsular polysaccharide conjugate is within the above range. The molecular weight of the compound is either

[0158] In certain embodiments, the GBS serotype VII capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0159] In certain embodiments, the GBS serotype VIII capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0160] In certain embodiments, the GBS serotype IX capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0161] In one embodiment, the conjugates of the invention have at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.97 or 0.98 mM sialic acid per mM polysaccharide. In a preferred embodiment, the conjugates have at least about 0.9 or 0.95 mM sialic acid per mM polysaccharide.

[0162] In certain embodiments, the GBS serotype IV capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0163] In certain embodiments, the GBS serotype Ia capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0164] In certain embodiments, the GBS serotype Ib capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0165] In certain embodiments, the GBS serotype II capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0166] In certain embodiments, the GBS serotype III capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0167] In certain embodiments, the GBS serotype V capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0168] In certain embodiments, the GBS serotype VI capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0169] In certain embodiments, the GBS serotype VII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0170] In certain embodiments, the GBS serotype VIII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0171] In certain embodiments, the GBS serotype IX capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0172] In certain embodiments, the conjugates of the invention comprise less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM repeating saccharide unit, hi other embodiments, the conjugates comprise at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM O-acetate per mM repeating saccharide unit.

[0173] In certain embodiments, the GBS serotype IV capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0174] In certain embodiments, the GBS serotype Ia capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0175] In certain embodiments, the GBS serotype Ib capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0176] In certain embodiments, the GBS serotype II capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0177] In certain embodiments, the GBS serotype III capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0178] In certain embodiments, the GBS serotype V capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0179] In certain embodiments, the GBS serotype VI capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0180] In certain embodiments, the GBS serotype VII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0181] In certain embodiments, the GBS serotype VIII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0182] In certain embodiments, the GBS serotype IX capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0183] In further embodiments, the immunogenic conjugate comprises less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free GBS capsular polysaccharide relative to the total amount of GBS capsular polysaccharide. In a preferred embodiment, the immunogenic conjugate comprises less than about 5% unreacted free saccharide relative to the total amount of GBS capsular polysaccharide.

[0184] In yet another embodiment, the ratio of GBS capsular polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.0. In one aspect, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.5 and about 2.0, between about 0.5 and about 1.5, between about 0.5 and about 1.0, between about 1.0 and about 1.5, or between about 1.0 and about 2.0. In a preferred embodiment, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.8 and about 1.0.

[0185] In another embodiment, the degree of conjugation of the conjugate 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 a preferred embodiment, the degree of conjugation of the conjugate is between 2 and 5.

[0186] Conjugation Conjugation can be direct, where an atom from the polysaccharide is covalently bonded to an atom from the protein surface, or alternatively, conjugation can be via a linker molecule, which reacts with both the polysaccharide and the protein and connects the two, tethering the carbohydrate to the protein.

[0187] When the carrier and one or more antigens, such as polysaccharides, are conjugated (i.e., covalently associated), the conjugation can be by any chemical method, process, or genetic technique known to those skilled in the art. For example, the carrier polypeptide and one or more antigens selected from the group including carbohydrates, oligosaccharides, lipids, lipooligosaccharides, polysaccharides, oligosaccharide-protein conjugates, polysaccharide-protein conjugates, peptide-protein conjugates, oligosaccharide-peptide conjugates, polysaccharide-peptide conjugates, protein-protein conjugates, lipooligosaccharide-protein conjugates, polysaccharide-protein conjugates, or any combination thereof, can be coupled to the carrier polypeptide by (1) direct coupling through protein functional groups (e.g., thiol-thiol bonds, amine-carboxyl bonds, amine-aldehyde bonds; enzymatic direct coupling), (2) homobifunctional coupling of amines (e.g., using bis-aldehydes), (3) homobifunctional coupling of thiols (e.g., bis-aldehydes), (4) homobifunctional coupling of thiols (e.g., bis-aldehydes), (5) homobifunctional coupling of thiols (e.g., bis-aldehydes), (6) homobifunctional coupling of thiols (e.g., bis-aldehydes), (7) homobifunctional coupling of thiols (e.g., bis-aldehydes), (8) homobifunctional coupling of thiols (e.g., bis-aldehydes), (9) homobifunctional coupling of thiols (e.g., bis-aldehydes), (10) homobifunctional coupling of thiols (e.g., bis-aldehydes), (11) homobifunctional coupling of thiols (e.g., bis-aldehydes), (12) homobifunctional coupling of thiols (e.g., bis-aldehydes), (13) homobifunctional coupling of thiols (e.g., bis-aldehydes), (14 -maleimides), (4) homobifunctional coupling via photoactivatable reagents, (5) heterobifunctional coupling of amines with thiols (e.g., using maleimides), (6) heterobifunctional coupling via photoactivatable reagents (e.g., β-carbonyl diazo family), (7) introduction of amine-reactive groups into polysaccharides or oligosaccharides via cyanogen bromide activation or carboxymethylation, (8) introduction of thiol-reactive groups into polysaccharides or oligosaccharides via heterobifunctional compounds such as maleimide-hydrazides, (9) protein-lipid conjugation via introduction of hydrophobic groups into proteins, and (10) protein-lipid conjugation via incorporation of reactive groups into lipids. Heterobifunctional "non-covalent coupling" techniques such as biotin-avidin interactions are also contemplated. Other methods known in the art for achieving conjugation of oligo- and polysaccharides to immunogenic carrier proteins are also within the scope of some embodiments of the present invention.

[0188] In one embodiment, the GBS capsular polysaccharide-protein conjugate is obtained by activating the polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide can be coupled to an amino group on a carrier protein directly or via a spacer (linker) group. For example, the spacer can be cystamine or cysteamine to obtain a thiolated polysaccharide that can be coupled 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, SIB, SIAB, sulfo-SIAB, SIA or SBAP).

[0189] In one embodiment, a cyanate ester (which may be made by CDAP chemistry) is coupled with hexanediamine or adipic dihydrazide (ADH), and an amino-derivatized sugar is conjugated to a carrier protein using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. Such conjugates are described, for example, in International Patent Application Publication Nos. WO93 / 15760, WO95 / 08348, and WO96 / 29094.

[0190] Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC and TSTU. Many are described in International Patent Application Publication No. WO98 / 42721. Conjugation may involve a carbonyl linker that can be formed by reaction of the free hydroxyl group of the sugar with 1,1 carbonyldiimidazole (CDI) or 1,1 carbonyldi-1,2,4 triazole (CDT) (see Bethell et al., J. Biol. Chem., 254:2572-2574 (1979); Hearn et al., J. Chromatogr., 218:509-518 (1981)), followed by reaction with the protein to form a carbamate bond. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI / CDT to form a CDI / CDT carbamate intermediate, and coupling the CDI / CDT carbamate intermediate to an amino group on the protein.

[0191] In a preferred embodiment, the GBS capsular polysaccharide-protein conjugates of the invention are prepared using reductive amination, which involves two steps: (1) oxidation of the polysaccharide to generate aldehyde functional groups from vicinal diols in individual hexasaccharide units, and (2) reduction of the activated polysaccharide and carrier protein to form the conjugate.

[0192] In one embodiment, the GBS capsular polysaccharide is (a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction by the addition of a quenching agent to provide activated GBS capsular polysaccharide. It is activated (oxidized) by a process that involves

[0193] In certain embodiments of the invention, the concentration of the isolated capsular polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL, such as between about 0.5 mg / mL and about 5.0 mg / mL, between about 1.0 mg / mL and about 3.0 mg / mL, or about 2.0 mg / mL.

[0194] In certain embodiments, the oxidizing agent is periodate. Periodate oxidizes adjacent hydroxyl groups to form carbonyl or aldehyde groups, resulting in the cleavage of CC bonds. The term "periodate" includes both periodate and periodic acid. The term also includes both metaperiodate (IO4-) and orthoperiodate (IO65-). The term "periodate" also includes various salts of periodate, including sodium periodate and potassium periodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In a preferred embodiment, the periodate used to oxidize GBS capsular polysaccharide is metaperiodate. In a preferred embodiment, the periodate used to oxidize serotype capsular polysaccharide is sodium metaperiodate.

[0195] In another embodiment, the polysaccharide is reacted with 0.01 to 10.0, 0.05 to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7 to 0.8, 0.05 to 0.5, or 0.1 to 0.3 molar equivalents of the oxidizing agent. In certain embodiments, the polysaccharide is reacted with about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95 molar equivalents of the oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.1 molar equivalents of the oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.15 molar equivalents of the oxidizing agent. In additional embodiments, the polysaccharide is reacted with about 0.15 molar equivalents of the oxidizing agent. In one embodiment, the polysaccharide is reacted with about 0.25 molar equivalents of the oxidizing agent. In yet another embodiment, the polysaccharide is reacted with about 0.5 molar equivalents of the oxidizing agent. In an alternative embodiment, the polysaccharide is reacted with about 0.6 molar equivalents of the oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.7 molar equivalents of the oxidizing agent.

[0196] In one embodiment of the invention, the duration of the oxidation reaction is between about 1 hour and about 50 hours, between about 10 hours and about 30 hours, between about 15 hours and about 20 hours, between about 15 hours and about 17 hours, or about 16 hours.

[0197] In another aspect of the invention, the temperature of the oxidation reaction is maintained between about 2° C. and about 25° C., between about 2° C. and about 8° C., or between about 20° C. and about 25° C. In one preferred embodiment, the temperature of the reaction is maintained at about 23° C. In another preferred embodiment, the temperature of the reaction is maintained at about 5° C.

[0198] In a further aspect, the oxidation reaction is carried out in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) and Bis-Tris. In a preferred embodiment, the buffer is potassium phosphate.

[0199] In additional embodiments, the buffer has a concentration of between about 1 mM and about 500 mM, between about 1 mM and about 300 mM, or between about 50 mM and about 200 mM. In a preferred embodiment, the buffer has a concentration of about 100 mM.

[0200] In one aspect, the oxidation reaction is carried out at a pH between about 4.0 and about 8.0, between about 5.0 and about 7.0, or between about 5.5 and about 6.5. In a preferred embodiment, the pH is about 6.0.

[0201] In one embodiment, the activated GBS capsular polysaccharide is obtained by reacting about 0.5 mg / L to about 5.0 mg / mL of isolated capsular polysaccharide with about 0.05 to about 0.3 molar equivalents of periodate at a temperature between about 20° C. and 25° C.

[0202] In another embodiment, the activated GBS capsular polysaccharide is obtained by reacting about 0.5 mg / L to about 5.0 mg / mL of isolated capsular polysaccharide with about 0.05 to about 0.3 molar equivalents of periodate at a temperature between about 2° C. and about 8° C.

[0203] In another embodiment, the activated GBS capsular polysaccharide is purified according to methods known to those skilled in the art, such as gel permeation chromatography (GPC), dialysis or ultrafiltration / hemodiafiltration, etc. For example, the activated capsular polysaccharide is purified by hemodiafiltration using a concentration and ultrafiltration device.

[0204] In one embodiment, the degree of oxidation of the activated GBS capsular polysaccharide is between 5 and 25, such as between 5 and 15, between 5 and 10, between 10 and 25, between 10 and 20, between 10 and 15, etc. In a preferred embodiment, the degree of oxidation of the activated GBS capsular polysaccharide is between 10 and 20, between 11 and 19, between 12 and 18, between 13 and 17, or between 14 and 16.

[0205] In another embodiment, the activated GBS capsular polysaccharide is between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 300 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 400 kDa, between about 300 kDa and about 5 ... In a preferred embodiment, the activated GBS capsular polysaccharide has a molecular weight of between about 75 kDa and about 400 kDa.

[0206] In one embodiment, the activated GBS capsular polysaccharide is lyophilized, optionally in the presence of a sugar. In a preferred embodiment, the sugar is selected from sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit. In a preferred embodiment, the sugar is sucrose. The lyophilized activated capsular polysaccharide can then be combined with a solution containing a carrier protein.

[0207] In another embodiment, the activated GBS capsular polysaccharide is combined with a carrier protein and lyophilized, optionally in the presence of a sugar. In one aspect, the sugar is selected from sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit. In a preferred embodiment, the sugar is sucrose. The co-lyophilized polysaccharide and carrier protein can then be resuspended in a solution and reacted with a reducing agent.

[0208] Activated GBS capsular polysaccharides are (a) combining activated GBS capsular polysaccharide with a carrier protein; (b) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; The antibody can be conjugated to a carrier protein by a process comprising:

[0209] Conjugation of activated GBS capsular polysaccharide to a protein carrier by reductive amination in a polar aprotic solvent is suitable for maintaining low levels of free polysaccharide, as compared, for example, to reductive amination in aqueous solution, where the level of unreacted (free) polysaccharide increases significantly. In a preferred embodiment, steps (a) and (b) are carried out in a polar aprotic solvent.

[0210] In one embodiment, step (a) comprises dissolving the lyophilized GBS capsular polysaccharide in a solution comprising the carrier protein and a polar aprotic solvent, hi another embodiment, step (a) comprises dissolving the co-lyophilized GBS capsular polysaccharide and carrier protein in a polar aprotic solvent.

[0211] In one embodiment, the polar aprotic solvent is selected from the group consisting of dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF) and hexamethylphosphoramide (HMPA). In a preferred embodiment, the polar aprotic solvent is DMSO.

[0212] When steps (a) and (b) are carried out in an aqueous solution, steps (a) and (b) are preferably carried out in a buffer in the aqueous medium at a pH between about 6.0 and about 8.5, between about 7.0 and about 8.0, or between about 7.0 and about 7.5, selected from PBS, MES, HEPES, Bis-Tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine, or HEPB. In a preferred embodiment, the buffer is PBS. In a preferred embodiment, the pH is about 7.3.

[0213] In one embodiment, the concentration of the activated GBS capsular polysaccharide in step (b) is between about 0.1 mg / mL and about 10.0 mg / mL, between about 0.5 mg / mL and about 5.0 mg / mL, or between about 0.5 mg / mL and about 2.0 mg / mL. In a preferred embodiment, the concentration of the activated serotype GBS capsular polysaccharide in step (b) is between about 0.1 mg / mL and about 10.0 mg / mL, between about 0.5 mg / mL and about 5.0 mg / mL, or between about 0.5 mg / mL and about 2.0 mg / mL. g / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2.0 mg / mL, about 2.1 mg / mL, about 2.2 mg / mL, about 2.3 mg / mL, about 2.4 mg / mL, about 2.5 mg / mL, about 2.6 mg / mL, about 2.7 mg / mL, about 2.8 mg / mL, about 2.9 mg / mL, or about 3.0 mg / mL.

[0214] In preferred embodiments, the initial ratio (weight / weight) of activated serotype GBS capsular polysaccharide to carrier protein is between 5:1 and 0.1:1, 2:1 and 0.1:1, 2:1 and 1:1, 1.5:1 and 1:1, 0.1:1 and 1:1, 0.3:1 and 1:1, 0.6:1 and 1:1. In preferred embodiments, the initial ratio of activated serotype GBS capsular polysaccharide to carrier protein is about 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1.

[0215] In certain embodiments, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc in the presence of a Bronsted or Lewis acid, an amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB), etc. In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0216] In another embodiment, the amount of reducing agent used in step (b) is between about 0.1 and about 10.0 molar equivalents, between about 0.5 and about 5.0 molar equivalents, or between about 1.0 and about 2.0 molar equivalents. In a preferred embodiment, the amount of reducing agent used in step (b) is about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 molar equivalents.

[0217] In a preferred embodiment, the duration of step (b) is between 1 and 60 hours, between 10 and 50 hours, between 40 and 50 hours, or between 42 and 46 hours. In a preferred embodiment, the duration of step (b) is about 44 hours.

[0218] In further embodiments, the temperature of the reaction in step (b) is maintained between 10° C. and 40° C., between 15° C. and 30° C., or between 20° C. and 26° C. In a preferred embodiment, the temperature of the reaction in step (b) is maintained at about 23° C.

[0219] In an additional embodiment, the process for the preparation of an immunogenic conjugate comprising GBS capsular polysaccharide covalently bound to a carrier protein further comprises a step of capping (quenching) unreacted aldehydes by the addition of borohydride (step (c)).

[0220] In one embodiment, the capping reagent is a borohydride selected from the group consisting of sodium borohydride (NaBH4), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride and magnesium borohydride. In a preferred embodiment, the capping reagent is sodium borohydride.

[0221] In yet another embodiment, the amount of borohydride used in step (c) is , between about 0.1 and about 10.0 molar equivalents, between about 0.5 and about 5.0 molar equivalents, or between about 1.0 and 3.0 molar equivalents. In a preferred embodiment, the amount of borohydride used in step (c) is about 2.0 molar equivalents.

[0222] In one preferred embodiment, the borohydride used in step (c) is NaBH4 at a concentration of about 2.0 molar equivalents.

[0223] In one embodiment, the duration of step (c) is between 0.1 and 10 hours, between 0.5 and 5 hours, between 2 and 4 hours. In a preferred embodiment, the duration of step (c) is about 3 hours.

[0224] In another embodiment, the temperature of the reaction in step (c) is maintained between about 15° C. and about 45° C., between about 15° C. and about 30° C., or between about 20° C. and about 26° C. In a preferred embodiment, the temperature of the reaction in step (c) is maintained at about 23° C.

[0225] After conjugation and capping of the GBS capsular polysaccharide with the carrier protein, the polysaccharide-protein conjugate can be purified (enriched in terms of the amount of polysaccharide-protein conjugate) by a variety of techniques known to those skilled in the art, including dialysis, concentration / hemodiafiltration operations, tangential flow filtration, precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography) and depth filtration.

[0226] In further embodiments, the immunogenic conjugate comprises less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free GBS capsular polysaccharide relative to the total amount of GBS capsular polysaccharide. In a preferred embodiment, the immunogenic conjugate comprises less than about 5% unreacted free saccharide relative to the total amount of GBS capsular polysaccharide.

[0227] In a preferred embodiment, the GBS polysaccharide-protein conjugate has a molecular weight between about 300 kDa and about 20,000 kDa, such as between about 1,000 kDa and about 15,000 kDa or between about 1,000 kDa and about 10,000 kDa.

[0228] In yet another embodiment, the ratio of GBS capsular polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.0. In one aspect, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.5 and about 2.0, between about 0.5 and about 1.5, between about 0.5 and about 1.0, between about 1.0 and about 1.5, or between about 1.0 and about 2.0. In a preferred embodiment, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.8 and about 1.0.

[0229] In another embodiment, the degree of conjugation of the conjugate 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 a preferred embodiment, the degree of conjugation of the conjugate is between 2 and 5.

[0230] In one embodiment of the present invention, the GBS capsular polysaccharide-protein conjugate is obtained by the reductive amination method described above. For example, in one embodiment, the present disclosure provides a method for the preparation of a GBS capsular polysaccharide-protein conjugate comprising: (a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction by the addition of a quenching agent to provide activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; and optionally (e) capping any unreacted aldehyde by addition of sodium borohydride (NaBH4); The present invention provides a GBS capsular polysaccharide-protein conjugate comprising a polysaccharide conjugated to a carrier protein, the polysaccharide being produced or obtainable by a method comprising the steps of:

[0231] In a preferred embodiment, steps (c) and (d) are carried out in DMSO.

[0232] In another aspect of the invention, the GBS capsular polysaccharide-protein conjugates of the invention are prepared using reductive amination as described above, except that 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) are used as co-oxidants in the activation / oxidation step. See International Patent Application Publication No. WO2014 / 097099, which is incorporated by reference in its entirety. In such an embodiment, the GBS capsular polysaccharide-derived glycoconjugates are prepared using TEMPO free radical to oxidize the primary alcohol of the sugar to an aldehyde using NCS as a co-oxidant (hereinafter "TEMPO / NCS oxidation"), as described in Example 7 and in International Patent Application Publication No. WO2014 / 097099. Thus, in one aspect, conjugates of GBS capsular polysaccharide are obtainable by a method comprising the steps of a) reacting GBS capsular polysaccharide with TEMPO and NCS in a solvent to produce an activated saccharide, and b) reacting the activated saccharide with a carrier protein containing one or more amine groups (hereinafter "TEMPO / NCS-reductive amination"). In one embodiment, the solvent may be an aqueous solvent or DMSO.

[0233] In one embodiment, the GBS capsular polysaccharide-protein conjugate is obtained by the method. For example, in one embodiment, the present disclosure provides a GBS capsular polysaccharide-protein conjugate comprising a polysaccharide conjugated to a carrier protein, which is produced or obtainable by a method comprising: a) reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in a solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amine groups. In one embodiment, the solvent may be an aqueous solvent or DMSO.

[0234] immunogenic composition After the individual conjugates are purified, they can be combined to formulate an immunogenic composition of the invention that can be used, for example, in a vaccine. Formulation of the immunogenic composition of the invention can be accomplished using art-recognized methods.

[0235] An "immune response" to an immunogenic composition is the development in a subject of a humoral and / or cell-mediated immune response to molecules (e.g., antigens such as proteins or polysaccharides) present in the composition of interest. For the purposes of this invention, a "humoral immune response" is an antibody-mediated immune response, involving the production of antibodies with affinity for the antigens present in the immunogenic composition of the invention, whereas a "cell-mediated immune response" is one mediated by T-lymphocytes and / or other white blood cells. A "cell-mediated immune response" is elicited by the presentation of antigen epitopes in association with class I or class II molecules of the major histocompatibility complex (MHC). This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic T lymphocyte cells (CTLs). CTLs are encoded by MHC or CD1 and are expressed by cells. CTLs have specificity for peptide or lipid antigens that are presented in association with proteins expressed on the surface of cells. CTLs help induce and promote the intracellular destruction of intracellular microorganisms, or the lysis of cells infected with such microorganisms. Another aspect of cell-mediated immunity involves antigen-specific responses by helper T cells. Helper T cells act to stimulate the function and help focus the activity of nonspecific effector cells against cells displaying peptide antigens in association with classical or nonclassical MHC molecules on their surface. "Cell-mediated immune response" also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other white blood cells, including those derived from CD4+ and CD8+ T cells. The ability of a particular antigen or composition to stimulate a cell-mediated immunological response can be determined by a number of assays, such as lymphocyte proliferation (lymphocyte activation) assays, CTL cytotoxicity cell assays, by assaying for T-lymphocytes specific for the antigen in sensitized subjects, or by measuring cytokine production by T cells in response to restimulation with antigen. Such assays are well known in the art, see, e.g., Erickson, A. L. et al., J. Immunol., 151(8):4189-4199 (1993); Doe, B. et al., Eur. J. Immunol., 24(10):2369-2376 (1994).

[0236] The term "immunogenicity" refers to the ability of an antigen or vaccine to elicit either a humoral or cell-mediated immune response, or both.

[0237] An "immunogenic amount" or an "immunologically effective amount" or a "dose", each of which are used interchangeably herein, generally refer to an amount of an antigen or immunogenic composition sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to those of skill in the art.

[0238] "Immune interference" or "significant immune interference", as used herein, refers to a statistically significant reduction in the immune response to an individual antigen in a multivalent or multicomponent vaccine compared to the immune response to the same antigen when administered in a monovalent vaccine.

[0239] A "protective" immune response refers to the ability of an immunogenic composition to elicit an immune response, either humoral or cell-mediated, that serves to protect a subject from infection. The protection provided need not be absolute, i.e., to completely prevent or eradicate infection, provided there is a statistically significant improvement compared to a control population of subjects, e.g., infected animals that have not been administered the vaccine or the immunogenic composition. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of infection. Several assays are known in the art for determining whether an immune response is indicative of a "protective immune response." For example, an increase in antibody levels may be measured by a binding assay, such as a whole cell ELISA assay, as described further below. Other assays include measuring functional antibody responses, such as the facilitation of bacterial killing, which may be tested with an opsonophagocytosis assay (OPA), as described below. In certain circumstances, a "protective immune response" may include eliciting a two-fold increase in antibody levels or a four-fold increase in antibody levels specific to a particular antigen in at least 50% of subjects. In other contexts, a "protective immune response" can encompass a reduction in bacterial counts of at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more.

[0240] The amount of a particular conjugate in a composition is generally calculated based on the total polysaccharide, conjugated and unconjugated, for that conjugate. For example, a GBS capsular polysaccharide conjugate having 20% ​​free polysaccharide will contain about 80 mcg / ml of conjugated GBS capsular polysaccharide in a 100 mcg / ml GBS capsular polysaccharide dose. The conjugate is expected to have about 0.01 mg / ml to about 100 mcg / ml of each polysaccharide, and about 20 mcg / ml of unconjugated GBS capsular polysaccharide. The contribution of the protein carrier to the conjugate is not usually taken into account when calculating the dose of the conjugate. The amount of conjugate may vary depending on the serotype of the streptococcus. In general, each dose will contain about 0.01 mg / ml to about 100 mcg / ml, particularly about 1 mcg / ml to about 70 mcg / ml, more preferably about 5 mcg / ml to about 50 mcg / ml of each polysaccharide. The "immunogenic amounts" of the different polysaccharide components in the immunogenic composition may vary, with each amount being about 0.01 mcg / ml, about 0.1 mcg / ml, about 0.25 mcg / ml, about 0.5 mcg / ml, about 1 mcg / ml, about 2 mcg / ml, about 3 mcg / ml, about 4 mcg / ml, about 5 mcg / ml, about 6 mcg / ml, about 7 mcg / ml, about 8 mcg / ml, about 9 mcg / ml, about 10 mcg / ml, about 11 mcg / ml, about 12 mcg / ml, about 13 mcg / ml, about 14 mcg / ml, about 15 mcg / ml, about 16 mcg / ml, about 17 mcg / ml, about 18 mcg / ml, about 19 mcg / ml, about 20 The polysaccharide antigen may comprise about 100mcg / ml, about 9mcg / ml, about 10mcg / ml, about 15mcg / ml, about 20mcg / ml, about 25mcg / ml, about 30mcg / ml, about 40mcg / ml, about 50mcg / ml, about 60mcg / ml, about 70mcg / ml, about 80mcg / ml, about 90mcg / ml, or about 100mcg / ml of any particular polysaccharide antigen. A dose or immunogenic amount of a multivalent immunogenic composition is expected to indicate the dose of each polysaccharide unless otherwise indicated. For example, a 10mcg / ml dose of a hexavalent immunogenic composition is expected to contain 10mcg / ml of each of the six polysaccharides.

[0241] The effectiveness of an antigen as an immunogen can be measured by measuring the level of circulating antibodies specific for the antigen in serum, by measuring the level of B cell activity, using immunoassays, immunoprecipitation assays, functional antibody assays, such as in vitro opsonization assays, and many other assays known in the art. Another measure of the effectiveness of an antigen as a T cell immunogen can be measured either by proliferation assays, by cytolytic assays, such as chromium release assays to measure the ability of T cells to lyse their specific target cells, and the like. Furthermore, in the present invention, an "immunogenic amount" may be defined by measuring the serum level of antigen-specific antibodies elicited after administration of the antigen, or by measuring the ability of the antibodies so elicited to enhance the opsonophagocytic ability of certain leukocytes as described herein. The level of protection of the immune response can be measured by inoculating an immunized host with the injected antigen. For example, if the antigen against which an immune response is desired is a bacterium, the level of protection elicited by an "immunogenic amount" of the antigen can be measured by detecting the percent survival or percent death after inoculation of an animal with bacterial cells. In one embodiment, the amount of protection can be measured by measuring at least one symptom associated with bacterial infection, such as fever associated with infection. The amount of each of the antigens in a multi-antigen or multi-component vaccine or immunogenic composition will vary with respect to each of the other components and can be determined by methods known to those skilled in the art. Such methods would include, for example, procedures for measuring immunogenicity and / or in vivo efficacy.

[0242] The term "immunogenic composition" refers to any pharmaceutical composition that contains an antigen, e.g., a microorganism, or a component thereof, and can be used to elicit an immune response in a subject. The immunogenic compositions of the present invention can be used to treat humans susceptible to GBS infections by administering the immunogenic compositions via systemic transdermal or mucosal routes. These administrations can include injection via intramuscular (im), intraperitoneal (ip), intradermal (id) or subcutaneous routes, application by a patch or other transdermal delivery device, or via mucosal administration to the oral / alimentary, respiratory or genitourinary tract. In one embodiment, the immunogenic compositions can be used in the manufacture of vaccines or in the elicitation of polyclonal or monoclonal antibodies that can be used to passively protect or treat animals.

[0243] In one aspect, the present invention provides a method for the treatment of a pulmonary artery disease comprising administering an effective amount of at least one of the compounds as described herein. The present invention relates to an immunogenic composition comprising at least one polysaccharide, oligosaccharide, polysaccharide-protein conjugate, or biological equivalent thereof. For example, in one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the capsular polysaccharide is selected from the group consisting of group B Streptococcus serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX, and the capsular polysaccharide has a sialic acid level of greater than about 60%. In another example, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharide from group B Streptococcus serotype IV and at least one additional serotype selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII, and IX. In another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotype IV and at least two additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In yet another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotype IV and at least three additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In a further embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotype IV and at least four additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In a particular embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from group B Streptococcus serotypes Ia, Ib, II, III and V. In another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from group B Streptococcus serotypes Ia, Ib, II, III and IV.In yet another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate comprising capsular polysaccharides from group B Streptococcus serotype IV and at least five additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In one such embodiment, the immunogenic composition comprises six polysaccharide-protein conjugates comprising capsular polysaccharides from group B Streptococcus serotypes Ia, Ib, II, III, IV and V.

[0244] In one embodiment, the immunogenic composition of the invention comprises from 2 to 10 different serotypes of S. agalactiae. Thus, in one embodiment, the immunogenic composition of the invention is a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-valent GBS conjugate composition. In one such embodiment, the immunogenic composition is a 5-valent GBS conjugate composition. In another embodiment, the immunogenic composition is a 6-valent GBS conjugate composition. In yet another embodiment, the immunogenic composition is a 7-valent GBS conjugate composition. In a further embodiment, the immunogenic composition is an 8-valent GBS conjugate composition.

[0245] Despite previous teachings of using fewer than six, fewer than five, or fewer than four GBS antigens in a composition (see International Patent Application Publication Nos. WO 2006 / 082527 and WO 2006 / 082530) and experience with immune interference, particularly with the use of serotype V in a multivalent composition (see International Patent Application Publication No. WO 2012 / 035519), the present invention does not demonstrate any significant immune interference with the use of four or more GBS antigens in a multivalent composition or with the use of serotype V. Thus, the present invention relates to multivalent immunogenic compositions comprising polysaccharide-protein conjugates comprising at least four GBS capsular polysaccharide serotypes, such as at least five GBS capsular polysaccharide serotypes, at least six GBS capsular polysaccharide serotypes, at least seven GBS capsular polysaccharide serotypes, at least eight GBS capsular polysaccharide serotypes, or at least nine GBS capsular polysaccharide serotypes, etc., wherein the composition does not have significant immune interference. In a specific embodiment, the immunogenic composition comprises GBS capsular polysaccharide serotype V.

[0246] The polysaccharide-protein conjugates may comprise the same or different protein carriers. In one embodiment, the conjugates comprise the same protein carrier, and the saccharides are conjugated to the same molecule of protein carrier (the carrier molecule having two or more different polysaccharides conjugated thereto) (see, for example, International Patent Application Publication No. WO2004 / 083251). In another embodiment, the polysaccharides are each independently conjugated to different molecules of protein carrier (each molecule of protein carrier having only one type of polysaccharide conjugated thereto). In said embodiment, the capsular saccharides are said to be independently conjugated to the carrier protein.

[0247] Optimal amounts of components for a particular immunogenic composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. After an initial vaccination, subjects can receive one or several booster immunizations at appropriately spaced intervals.

[0248] The immunogenic composition of the present invention may further comprise one or more preservatives in addition to the multiple capsular polysaccharide protein conjugates. The FDA requires that biological products in multi-dose (multi-dose) vials contain a preservative, with few exceptions. The present invention contemplates the use of such multi-dose vials. Vaccine products that contain preservatives include vaccines that contain benzethonium chloride (anthrax), 2-phenoxyethanol (DTaP, HepA, Lyme, Polio (parenteral)) and phenol (pneumonia, typhoid (parenteral)). Preservatives approved for use in injectables include, for example, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol and phenylmercuric nitrate.

[0249] In another aspect, the invention relates to a composition including at least one of any of the polysaccharides described herein and a pharma- ceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic surfactant, inhibitor of free radical oxidation, diluent or carrier, or a mixture thereof.

[0250] The immunogenic composition may include one or more physiologically acceptable buffers selected from, but not limited to, HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. In a preferred embodiment, the buffer is histidine.

[0251] In one embodiment, the immunogenic composition comprises a buffer at a concentration of about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, or about 15 mM to about 20 mM. In a preferred embodiment, the immunogenic composition comprises a buffer at a concentration of about 10 mM to about 25 mM, most preferably about 20 mM.

[0252] In one preferred embodiment, the immunogenic composition comprises histidine at a concentration of about 20 mM.

[0253] In certain embodiments, the formulation has a solubility of about 5.0 to about 7.1, e.g., about 5.3 to about 7.1, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.0 to about 6.5, about 6 The formulation is buffered to a pH range of about 6.0 to about 7.0, such as about 6.3 to about 7.0, or about 6.5 to about 7.0. In another embodiment, the formulation is buffered to a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In a preferred embodiment, the formulation is buffered to a pH range of about 6.0 to about 7.0, most preferably about 6.5.

[0254] The immunogenic composition may comprise one or more non-ionic surfactants, including but not limited to polyoxyethylene sorbitan fatty acid esters, polysorbate-80 (Tween 80), polysorbate-60 (Tween 60), polysorbate-40 (Tween 40), polysorbate-20 (Tween 20), and polyoxyethylene alkyl ethers, including but not limited to BRIJ58, BRIJ35, and others, such as Triton X-100, Triton X-114, NP40, Span 85, and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121). In one embodiment, the immunogenic composition comprises polysorbate-80 or polysorbate-40, preferably polysorbate-80 (PS80).

[0255] In one embodiment, the immunogenic composition comprises a surfactant at about 0.001% to about 2% (v / w), about 0.001% to about 1%, about 0.001% to about 0.5%, about 0.001% to about 0.1%, about 0.001% to about 0.05%, about 0.001% to about 0.01%, about 0.001% to 0.005%, about 0.005% to about 2%, about 0.005% to about 1%, about 0.005% to about 0.5%, about 0.005% to about 0.1%, about 0.005% to about 0.05%, about 0.005% to about 0.01%, about 0.01% to about 2%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.01% to about 0.1%, about 0.01% to about 0.05%, about 0.01% to about 0.04%, about 0.01% to about 0. .03%, about 0.015% to about 2%, about 0.015% to about 1%, about 0.015% to about 0.5%, about 0.015% to about 0.1%, about 0.015% to about 0.05%, about 0.015% to about 0.04%, about 0.015% to about 0.03%, about 0.02% to about 2%, about 0.02% to about 1%, about 0.02% to about 0.5%, about 0.02% to about 0. In a preferred embodiment, the immunogenic composition comprises a surfactant at a concentration of about 0.01% to 0.03%, about 0.02% to about 0.05%, about 0.02% to about 0.04%, about 0.02% to about 0.03%, about 0.05% to about 2%, about 0.05% to about 1%, about 0.05% to about 0.5%, about 0.05% to about 0.1%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.1% to about 0.5%, or about 0.1% to 0.25%. In a preferred embodiment, the immunogenic composition comprises a surfactant at a concentration of about 0.01% to 0.03%, most preferably about 0.02%.

[0256] In another embodiment, the immunogenic composition comprises polysorbate-80 at a concentration of about 0.001% to about 2%, preferably up to about 0.25%, or polysorbate-40 at a concentration of about 0.001% to 1%, preferably up to about 0.5%.

[0257] In a preferred embodiment, the immunogenic composition comprises PS80 at a concentration of about 0.02%.

[0258] Pharmaceutically acceptable carriers should not be confused with "carrier proteins" that are used in binding the carbohydrates of the present invention to proteins and modify the immune response to the carbohydrates. To avoid confusion with the protein carriers described herein, the term pharma- ceutically acceptable diluent is preferred over pharma-ceutically acceptable carrier, although these terms may sometimes be used interchangeably. The term "pharma-ceutically acceptable carrier" refers to a carrier approved by a federal regulatory agency, a state government or other regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, including humans and non-human mammals. The term "carrier" refers to a pharmaceutical composition. It refers to a diluent, adjuvant, excipient or vehicle with which something is administered. Suitable pharmaceutically acceptable diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Such pharmaceutically acceptable diluents can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, water for injection (WFI), sterile isotonic saline, phosphate buffer solution, adjuvant suspension, aqueous dextrose and glycerol solution, and combinations thereof can be used as liquid carriers, particularly for injectable solutions. Pharmaceutically acceptable diluents can further include small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance shelf life or effectiveness in the body. The preparation and use of pharmaceutically acceptable diluents are well known in the art. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. In one embodiment, the diluent is water, water for injection (WFI), adjuvant suspension or saline.In a particular embodiment, the diluent is any of the adjuvant suspensions described herein.In a preferred embodiment, the diluent is an aluminum-based adjuvant suspension, such as an aluminum phosphate suspension.

[0259] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone flour, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. In a preferred embodiment, the excipient is NaCl.

[0260] In one embodiment, the immunogenic composition comprises an excipient at a concentration of about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 10 mM to about 50 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM , about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 150 mM, about 20 mM to about 100 mM, about 20 mM to about 50 mM, about 20 mM to about 30 mM, 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 150 mM to about 500 mM, about 150 mM to about 450 mM, about 150 mM to about 400 mM, about 150 mM to about 350 mM, about 150 mM to about 300 mM, about 150 mM to about 250 mM, about 150 mM to about 200 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 40 0 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 500 mM,In a preferred embodiment, the immunogenic composition comprises an excipient, The agent is included at a concentration of from about 10 mM to about 250 mM, most preferably about 150 mM.

[0261] In one preferred embodiment, the excipient is NaCl at a concentration of about 150 mM.

[0262] The compositions can also contain minor amounts of wetting, bulking, emulsifying agents or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, lyophilized powders or cakes, etc. The formulation should be suitable for the mode of administration. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the immunogenic compositions of the present invention is contemplated.

[0263] In one embodiment, the immunogenic composition is lyophilized, optionally in the presence of at least one excipient. In a preferred embodiment, the at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol. In a preferred embodiment, the at least one excipient is selected from the group consisting of sucrose, mannitol and glycine. In a particular embodiment, the at least one excipient is sucrose. In another embodiment, the lyophilized composition comprises an additional excipient. In one such embodiment, the additional excipient is mannitol or glycine.

[0264] In another embodiment, the lyophilized composition comprises at least one sugar at about 1% (w / v) to about 10% (w / v), such as about 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10.0%. In a preferred embodiment, the lyophilized composition comprises at least one excipient at more than about 5.5% (w / v), such as more than about 7.0% (w / v). In further embodiments, the lyophilized composition comprises about 1% (w / v) to about 10% (w / v) of an additional excipient, such as about 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10.0%, etc. In a preferred embodiment, the lyophilized composition comprises about 1% (w / v) to about 10% (w / v) of at least one excipient and about 1% (w / v) to about 10% (w / v) of an additional excipient.

[0265] In yet another embodiment, the lyophilized composition is reconstituted with water, water for injection (WFI), an adjuvant suspension or saline. In a preferred embodiment, the diluent is an aluminum-based adjuvant suspension, such as an aluminum phosphate suspension.

[0266] In one embodiment, the composition comprises the isolated polysaccharide and carrier molecule described herein.Suitable carrier molecules may comprise proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles.Examples of particulate carriers include those derived from polymethylmethacrylate polymers, and microparticles derived from poly(lactide) and poly(lactide-co-glycolide), known as PLG.

[0267] The immunogenic compositions of the invention can further comprise one or more additional "immunomodulatory agents," which are agents that perturb or modify the immune system, such that either upregulation or downregulation of humoral and / or cell-mediated immunity is observed. In embodiments, upregulation of humoral and / or cell-mediated arm of immune system is preferred.Examples of certain immunomodulatory agents include adjuvants or cytokines, such as those described in U.S. Patent No. 5,254,339, or ISCOMATRIX (CSL Limited, Parkville, Australia).The term "adjuvant" refers to a compound or mixture that enhances the immune response to antigen, as further described herein.

[0268] Non-limiting examples of adjuvants that can be used in the compositions of the invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.); mineral gels, such as aluminum hydroxide gel; water-in-oil emulsions, such as Freund's complete and incomplete adjuvants; block copolymers (CytRx, Atlanta Ga.); SAF-M (Chiron, Emeryville, Calif.); AMPHIGEN® adjuvant; saponin; Quil A or other saponin fractions; monophosphoryl lipid A; and avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful as adjuvants in the vaccines of the invention include MF59 (U.S. Pat. No. 6,299,884) (containing 5% squalene, 0.5% polysorbate 80, and 0.5% Span 85 (which may contain various amounts of MTP-PE) formulated into submicron particles using a microfluidizer, such as the Model 110Y Microfluidizer (Microfluidics, Newton, Mass.)), and SAF (microfluidized into a submicron emulsion). containing 10% squalene, 0.4% polysorbate 80, 5% Pluronic block polymer L121 and thr-MDP, either mixed or vortexed to produce larger particle size emulsions; modified SEAM62 (5% (v / v) squalene (Sigma), 1% (v / v) SPAN® 85 surfactant (ICI surfactant), 0.7% (v / v) polysorbate 80 surfactant (ICI surfactant), 2.5% (v / v) ethanol, 200 μg / ml Quil A, containing 100 μg / ml cholesterol and 0.5% (v / v) lecithin); and modified SEAM 1 / 2, containing 5% (v / v) squalene, 1% (v / v) SPAN® 85 surfactant, 0.7% (v / v) polysorbate 80 surfactant, 2.5% (v / v) ethanol, 100 μg / ml Quil A and 50 μg / ml cholesterol.

[0269] Suitable adjuvants used to enhance immune responses further include, but are not limited to, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT), described in U.S. Patent No. 4,912,094. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGPs), available from Corixa (Hamilton, MT), and described in U.S. Patent No. 6,113,918, or derivatives or analogs thereof. One such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxy-tetrade-canoyl)]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-bD-glucopyranoside, also known as 529 (formerly known as RC529). The 529 adjuvant is formulated as an aqueous form (AF) or as a stable emulsion (SE).

[0270] Other adjuvants include cyclodextrin derivatives (U.S. Pat. No. 6,165,995); polyanionic polymers (U.S. Pat. No. 6,610,310); muramyl peptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) and N-acetyl-normuramyl-L-alanine-2-(1'-2' dipalmitoyl) ( ... 1,326,634); IC-31 (Intercell 1,326,634); Amphigen; Avridine; L121 / Squalene; D-Lactide-Polylactide / Glycoside; Pluronic Polyols; Killed Bordetella; Saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, Mass.), described in U.S. Pat. No. 5,057,540; Mycobacterium tuberculosis; Bacterial lipopolysaccharides; Synthetic polynucleotides, such as oligonucleotides containing CpG motifs (e.g., U.S. Pat. No. 6,207,646); IC-31 (Intercell 1,326,634), described in European Patents Nos. 1,296,713 and 1,326,634. AG, Vienna, Austria); pertussis toxin (PT) or a mutant thereof, cholera toxin or a mutant thereof (e.g., U.S. Pat. Nos. 7,285,281, 7,332,174, 7,361,355 and 7,384,640); or E. coli heat-labile toxin (LT) or a mutant thereof, in particular LT-K63, LT-R72 (e.g., U.S. Pat. Nos. 6,149,919, 7,115,730 and 7,291,588).

[0271] Other "immunomodulatory agents" that may be included in the vaccine include, for example, one or more of interleukin 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and mutant forms thereof); interferon-α, β and γ; granulocyte-macrophage colony-stimulating factor (GM-CSF) (see, e.g., U.S. Pat. No. 5,078,996 and ATCC Accession No. 39900); macrophage colony-stimulating factor (M-CSF); granulocyte colony-stimulating factor (G-CSF); or tumor necrosis factors α and β. Still other adjuvants useful in the immunogenic compositions described herein include chemokines, including, but not limited to, MCP-1, MIP-1α, MIP-1β, and RANTES; adhesion molecules, such as selectins, such as L-selectin, P-selectin, and E-selectin; mucin-like molecules, such as CD34, GlyCAM-1, and MadCAM-1; members of the integrin family, such as LFA-1, VLA-1, Mac-1, and p150.95; members of the immunoglobulin superfamily, such as PECAM, ICAMs, such as such as ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3; costimulatory molecules such as B7-1, B7-2, CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2 and DR6; and caspases (ICE).

[0272] It should be understood that the decision to use or not use an immunomodulatory agent and / or adjuvant, or the choice of which immunomodulatory agent and / or adjuvant to use, is expected to depend on the subject to which the vaccine or immunogenic composition is administered, the route of injection, and the number of injections to be given. For example, if the subject has been naturally exposed to a pathogen, an adjuvant may not be required because the vaccine antigen can effectively induce a memory response. In certain embodiments, the immunogenic composition will include one or more adjuvants. In one embodiment, the immunogenic composition includes an aluminum-based adjuvant. In one such embodiment, the aluminum adjuvant is aluminum hydroxide, aluminum phosphate, or aluminum hydroxyl phosphate. In certain embodiments, the adjuvant is aluminum phosphate. In another embodiment of the present invention, the immunogenic composition includes QS-21 as an adjuvant.

[0273] In one embodiment, the immunogenic composition comprises an adjuvant at a concentration of about 0.1 mg / ml to about 1.0 mg / ml, 0.1 mg / ml to about 0.9 mg / ml, 0.1 mg / ml to about 0.8 mg / ml, 0.1 mg / ml to about 0.7 mg / ml, 0.1 mg / ml to about 0.6 mg / ml, 0.1 mg / ml to about 0.5 mg / ml, 0.1 mg / ml to about 0.4 mg / ml, 0.1 mg / ml to about 0.3 mg / ml, 0.1 mg / ml to about 0.2 mg / ml, 0.25 mg / ml to about 0.95 mg / ml, 0.25 mg / ml to about 0.85 mg / ml, 0.25 mg / ml to about 0.75 mg / ml, 0.25 mg / ml to about 0.65 mg / ml, 0.25 mg / ml to about 0.5 mg / ml, 0.25 mg / ml to about 0.65 mg / ml, 0.25 mg / ml to about 0.75 mg / ml, 0.25 mg / ml to about 0.8 ... In some embodiments, the present invention is directed to a method for treating or preventing osteoporosis, comprising administering to a subject an effective amount of the present invention at a concentration ranging from about 0.55 mg / ml, 0.25 mg / ml to about 0.45 mg / ml, 0.25 mg / ml to about 0.35 mg / ml, 0.5 mg / ml to about 1.0 mg / ml, 0.5 mg / ml to about 0.9 mg / ml, 0.5 mg / ml to about 0.8 mg / ml, 0.5 mg / ml to about 0.75 mg / ml, 0.5 mg / ml to about 0.7 mg / ml, 0.5 mg / ml to about 0.65 mg / ml, 0.5 mg / ml to about 0.6 mg / ml, 0.75 mg / ml to about 1.0 mg / ml, 0.75 mg / ml to about 0.95 mg / ml, 0.75 mg / ml to about 0.9 mg / ml, and 0.75 mg / ml to about 0.85 mg / ml. In a preferred embodiment, the immunogenic composition comprises an adjuvant at a concentration of about 0.25 mg / ml to about 0.75 mg / ml, most preferably about 0.5 mg / ml.

[0274] In a preferred embodiment, the adjuvant is aluminum-based at a concentration of about 0.5 mg / ml, hi one such embodiment, the aluminum-based adjuvant is aluminum phosphate or aluminum hydroxyl phosphate.

[0275] In one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate as described herein, a buffer, a surfactant, an excipient, and optionally an adjuvant, and the composition is buffered to a pH of about 6.0 to about 7.0.

[0276] In one such embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the composition is buffered to a pH of about 6.0 to about 7.0, and the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0277] In one particular embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the composition is buffered to a pH of about 6.0 to about 7.0, and the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0278] In a preferred embodiment, the immunogenic composition comprises about 5 mcg / ml to about 50 mcg / ml of GBS polysaccharide-protein conjugate, about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0279] In one such embodiment, the immunogenic composition comprises at least two GBS polysaccharide-protein conjugates, a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the composition is buffered to a pH of about 6.0 to about 7.0, and the conjugates comprise capsular polysaccharides from Group B Streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

[0280] In one particular embodiment, the immunogenic composition comprises at least two GBS polysaccharide-protein conjugates, histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the composition is buffered to a pH of about 6.0 to about 7.0, and the conjugates comprise capsular polysaccharides from Group B Streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

[0281] In a preferred embodiment, the immunogenic composition comprises about 5 mcg / ml to about 50 mcg / ml each of at least two GBS polysaccharide-protein conjugates, about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate, wherein the conjugates comprise capsular polysaccharides from Group B Streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

[0282] Evaluation of immunogenic compositions Various in vitro tests are used to assess the immunogenicity of the immunogenic compositions of the present invention. For example, an in vitro opsonization assay is performed by incubating together a mixture of streptococcal cells, heat-inactivated serum containing specific antibodies against the antigen of interest, and an exogenous complement source. Opsonophagocytosis proceeds during incubation of the antibody / complement / streptococcal cell mixture with freshly isolated polymorphonuclear cells (PMN) or differentiated effector cells such as HL60. Antibody and complement coated bacterial cells are killed during opsonophagocytosis. The viable bacterial colony forming units (cfu) recovered from opsonophagocytosis are determined by plating the assay mixture. Titers are reported as the reciprocal of the highest dilution that gives 50% bacterial killing as determined by comparison with an assay control.

[0283] Whole cell ELISA assays may be used to assess in vitro immunogenicity and surface exposure of antigens, where the bacterial strain of interest (S. agalactiae) is coated onto a plate, such as a 96-well plate, and test sera from immunized animals are allowed to react with the bacterial cells. Antibodies specific for the test antigen, if reactive with a surface-exposed epitope of the antigen, can be detected by standard methods known to those of skill in the art. Alternatively, flow cytometry may be used to measure surface exposure of capsular polysaccharide antigens and specificity of antibodies, including monoclonal antibodies.

[0284] Antigens demonstrating the desired in vitro activity may then be tested in an in vivo animal challenge model. In certain embodiments, the immunogenic compositions are used in immunizing animals (e.g., mice) by methods and routes of immunization known to those of skill in the art (e.g., intranasal, parenteral, oral, rectal, intravaginal, transdermal, intraperitoneal, intravenous, subcutaneous, etc.). After immunization of animals with the GBS immunogenic compositions, the animals are challenged with a Streptococcus agalactiae strain and assayed for resistance to streptococcal infection.

[0285] In one embodiment, pathogen-free mice are immunized and inoculated with S. agalactiae. For example, mice are immunized with one or more doses of desired antigen in immunogenic composition. Then, mice are inoculated with S. agalactiae and survival is monitored over time after inoculation.

[0286] How to use "Immunocompromised" as used herein refers to a subject suffering from a deficiency in the cellular and / or humoral arms of the immune system. Thus, degrees of deficiency in immune function ranging from slight impairment in immune processes to complete immunosuppression are contemplated.

[0287] The term "subject" refers to a mammal, bird, fish, reptile, or any other animal. The term "subject" also includes humans. The term "subject" also includes household pets. Non-limiting examples of household pets include dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, birds, snakes, lizards, fish, turtles, and frogs. The term "subject" also includes livestock animals. Non-limiting examples of livestock animals include alpacas, bison, camels, cows, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

[0288] As used herein, "treatment" (including variations thereof, such as "treat" or "treated") refers to any one or more of the following: (i) prevention of infection or reinfection, such as traditional vaccines; (ii) reduction in severity or elimination of symptoms; and (iii) substantial or complete elimination of the pathogen or disorder in question. Thus, treatment can be accomplished prophylactically (before infection) or therapeutically (after infection). Prophylactic or therapeutic treatments can be used in the present invention. According to certain embodiments of the present invention, compositions and methods are provided that include prophylactically and / or therapeutically immunizing and treating a host animal against microbial infections (e.g., bacteria such as S. agalactiae). The methods of the present invention are useful for conferring prophylactic and / or therapeutic immunity to a subject. The methods of the present invention can also be performed in subjects for biomedical research applications.

[0289] In another aspect, the invention relates to a method of inducing an immune response against GBS in a subject by administering to the subject an effective amount of the immunogenic composition described herein. In one embodiment, the invention relates to a method of preventing or reducing a disease or condition associated with group B streptococcus in a subject by administering to the subject an effective amount of the immunogenic composition described herein. In an aspect, the invention relates to an immunogenic composition described herein for use as a medicament. In an aspect, the invention relates to an immunogenic composition described herein for use in a method of inducing an immune response against GBS in a subject. In a particular embodiment, the subject is a woman planning a pregnancy or a pregnant woman. In one such embodiment, the pregnant woman is in late pregnancy, such as at least 20 weeks or at least 27 weeks of pregnancy. In a preferred embodiment, the pregnant woman is between 27 and 36 weeks of pregnancy. In another embodiment, the subject is an elderly person, such as an adult aged 50 years or older, 65 years or older, and 85 years or older. In a further embodiment, the subject is immunocompromised. In one aspect, the subject may have a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease. In a preferred embodiment, the group B streptococcus is S. agalactiae.

[0290] In one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate comprising GBS serotype IV and at least one additional serotype selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In another embodiment, the conjugate comprises GBS serotype IV and at least two additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In an additional embodiment, the conjugate comprises GBS serotype IV and at least three additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In yet another embodiment, The conjugate comprises GBS serotype IV and at least four additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In a particular embodiment, the conjugate comprises GBS serotypes Ia, Ib, II, III and IV. In a further embodiment, the conjugate comprises GBS serotype IV and at least five additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In a further embodiment, the composition comprises GBS serotype V. In a particular embodiment, the conjugate comprises GBS serotypes Ia, Ib, II, III and V. In a preferred embodiment, the immunogenic composition comprises six polysaccharide-protein conjugates from GBS serotypes Ia, Ib, II, III, IV and V. One aspect of the present invention relates to an immunogenic composition that does not have immune interference.

[0291] The immunogenicity or effective amount of the immunogenic composition can be determined by performing a dose-response study in which subjects are immunized with increasing amounts of the immunogenic composition and the immune response is analyzed to determine the optimal dosage. The starting point of the study can be extrapolated from immunization data in animal models. The dosage can vary depending on the specific condition of the individual. The amount can be determined by routine testing using means known to those skilled in the art.

[0292] The immunogenic composition is administered to the subject in an immunologically effective amount of an appropriate dose to induce an immune response. The dosage may vary according to the specific conditions of the individual, such as age and weight. This amount can be determined by routine testing using means known to those skilled in the art.

[0293] In one embodiment, patients administered the immunogenic composition of the invention show a reduction in S. agalactiae carriage rates. Such a reduction in carriage or an extension of the time interval spent as a non-carrier after administration of the immunogenic composition is significant in terms of medical need. For example, a reduction in overall S. agalactiae carriage in carriers can be assessed after one dose of the immunogenic composition of the invention. For example, a group of adults aged 18-50 years can be screened for carriage by nasal, throat, axillary, rectal, perineal and vaginal swabs one day prior to administration of the immunogenic composition, followed by culture to determine carriage status. The group can then be administered the immunogenic composition of the invention, and one group can receive a control. After administration of the immunogenic composition, nasal, throat, axillary, rectal, perineal and vaginal swabs are performed weekly for 12 weeks and monthly for up to 6 months and compared to a placebo. One primary endpoint will be to compare carriage rates in patients following administration of the immunogenic composition with placebo at three-month intervals following immunization.

[0294] antibody An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless the context indicates otherwise, the term is intended to encompass intact polyclonal or monoclonal antibodies, as well as engineered antibodies (e.g., chimeric, humanized and / or derivatized to modify effector functions, stability and other biological activities) and fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chain (ScFv) and domain antibodies, including shark and camel antibodies), and fusion proteins comprising antibody portions, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity) and antibody fragments as described herein, as well as any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site. An antibody includes antibodies of any class, e.g., IgG, IgA, or IgM, etc. (or subclasses thereof), and an antibody need not be of any particular class. Depending on the amino acid sequence of the antibody in the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 in humans. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0295] An "antibody fragment" comprises only a portion of an intact antibody, which portion preferably retains at least one, and preferably most or all, of the functions normally associated with that portion when present in the intact antibody.

[0296] The "functional activity" of an antibody or a "functional antibody" as used herein refers to an antibody that can at a minimum specifically bind to an antigen. Additional functions are known in the art and may include additional components of the immune system that achieve pathogen clearance or killing, such as through opsonization, ADCC, or complement-mediated cytotoxicity. After antigen binding, any subsequent antibody function may be mediated through the Fc region of the antibody. The antibody opsonophagocytosis assay (OPA) is an in vitro assay designed to measure in vitro Ig complement-assisted killing of bacteria by effector cells (white blood cells), thus mimicking a biological process. Antibody binding may also directly inhibit the biological function of the antigen to which it is bound. In some embodiments, a "functional antibody" refers to an antibody that is functional as measured by bacterial killing in animal efficacy models or opsonophagocytic killing assays that demonstrate that the antibody kills bacteria.

[0297] In one aspect, the invention relates to an isolated antibody or fragment thereof that specifically binds to the polysaccharide described herein. An "isolated" antibody, as used herein, refers to an antibody that has been identified, separated and / or recovered from components of its natural environment. Contaminating components of its natural environment are materials that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In exemplary embodiments, the antibody will be purified (1) to 95% by weight of the antibody, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the antibody's natural environment is expected to be absent. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0298] An antibody that "specifically binds to" or is "specific for" a particular polysaccharide or an epitope on a particular polysaccharide is one that binds to the particular polysaccharide or an epitope on the particular polysaccharide without specifically binding to any other polysaccharide or polysaccharide epitope.

[0299] The term "label," as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. The label may be itself detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0300] The present invention further provides antibodies and antibody compositions that specifically and selectively bind to one or more antigens of the immunogenic compositions of the invention. In some embodiments, antibodies are produced upon administration of the immunogenic compositions of the invention to a subject. The present invention provides purified or isolated antibodies directed against one or more of the antigens of the immunogenic compositions of the present invention. In some embodiments, the antibodies of the present invention are functional as measured by killing bacteria in any animal efficacy model or via opsonophagocytic killing assay. In some embodiments, the antibodies of the present invention confer passive immunity to a subject. The present invention further provides polynucleotide molecules encoding the antibodies or antibody fragments of the present invention, as well as cells or cell lines (such as hybridoma cells or other engineered cell lines for recombinant production of antibodies) and transgenic animals that produce the antibodies or antibody compositions of the present invention using techniques well known to those skilled in the art.

[0301] The antibodies or antibody compositions of the invention may be used in a method of treating or preventing a streptococcal infection, disease or condition associated with S. agalactiae in a subject, comprising producing a polyclonal or monoclonal antibody preparation and using said antibody or antibody composition to confer passive immunity to the subject. The antibodies of the invention may also be useful in diagnostic methods, e.g., for detecting the presence or quantifying the level of one or more antigens of the immunogenic compositions of the invention.

[0302] The antibody response to repeating structures such as the polysaccharides of the present invention may exhibit some unique features. For example, the regularity of the repeating units may mean that antigen molecules of very different molecular weights can bind to antibodies specific for the polysaccharide. The second repeating structure of the longer polysaccharide can induce a T cell-independent antibody response. Thus, when using polysaccharides conjugated to protein carriers with T cell helper epitopes, both T cell-independent and T cell-dependent antibody responses can be stimulated. Thus, the immune response can be modified by the appropriate selection of polysaccharide size, regardless of whether a carrier protein is used or not.

[0303] Polyclonal antibodies In certain embodiments, the anti-polysaccharide antibody is a polyclonal antibody.Polyclonal antibody as defined herein refers to a mixture of antibodies with different specificities that are derived from serum preparations and originate from different B cell clones.The preparation and characterization of polyclonal antibodies are known in the art.

[0304] Polyclonal antibodies are raised in a subject, e.g., a mammal, by administering one or more injections of the immunogen or immunogenic composition described herein, as well as adjuvants, buffers and / or diluents, if desired. A range of animal species may be useful for the production of specific antisera. Typically, animals used for the production of anti-saccharide polyclonal antisera are non-human primates, goats, sheep, rabbits, mice, rats, hamsters or guinea pigs. Typically, the immunogen or immunogenic composition is injected into the mammal by multiple injections, with or without an adjuvant. The immunogenic material may include polysaccharides, oligosaccharides, polysaccharides, polysaccharide-protein conjugates described herein, or larger aggregates of immunogens. Typically, blood is collected from the immunized animal for the first 2-6 weeks after the initial immunization, allowed to clot, and serum is collected. The serum contains anti-saccharide polyclonal antibodies from the immunized animal and is often referred to as antiserum.

[0305] Monoclonal antibodies Anti-saccharide monoclonal antibodies may be prepared through the use of known hybridoma technology. Typically, preparing monoclonal antibodies involves first immunizing a suitable target animal host with a selected immunogen, including a polysaccharide, oligosaccharide, polysaccharide, or polysaccharide-protein conjugate of the invention. If desired, adjuvants, buffers, and / or diluents may be added. Immunization may be performed in a manner sufficient to induce B lymphocytes to produce or express antibodies that specifically bind to the polysaccharide or conjugate thereof. Alternatively, lymphocytes are immunized in vitro.

[0306] The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. The origin of the lymphocytes determines whether the monoclonal antibodies are of human or animal origin. Generally, peripheral blood lymphocytes ("PBL") are used when antibodies and cells of human origin are desired, and spleen cells or lymph node cells are used when non-human mammalian sources are desired.

[0307] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. Hybridoma cells are cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will typically contain hypoxanthine, aminopterin and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0308] The immortalized cell line is selected based on practical considerations such as species of origin, fusion and growth characteristics. For example, a suitable immortalized cell line is one that fuses efficiently, supports stable high level expression of antibody by the selected antibody-producing cells, and is sensitive to a medium such as HAT medium. Examples of immortalized cell lines include murine myeloma lines. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

[0309] The monoclonal antibodies are secreted into the culture medium by the hybridoma cells. The culture medium is then assayed for the presence of monoclonal antibodies that recognize and bind to polysaccharides. The anti-polysaccharide binding specificity of a particular monoclonal antibody produced by the hybridoma cells is determined by one of a number of procedures well known to those skilled in the art. For example, the antibody binding specificity can be determined by immunoprecipitation, radioimmunoassay (RIA), Western blot, enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (e.g., Biacore). The exact epitope recognized by the monoclonal antibody is determined by epitope mapping. Such techniques and assays are well known in the art.

[0310] After hybridoma cells producing antibodies with the desired specificity are identified, the clones are subcloned by limiting dilution and cultured using standard methods. Culture media suitable for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells are grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones are isolated and purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as, for example, protein A Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0311] Alternatively, antibodies from a desired species source with the desired specificity can be obtained through the use of phage display libraries. In addition, examples of methods and reagents that are particularly suitable for use in producing and screening antibody display libraries can be found in the art.

[0312] Use of antibodies In one aspect, the present invention relates to the use of the immunogenic compositions described herein to generate GBS antibodies and / or antibody fragments. The polysaccharide-protein conjugates described herein and / or antibodies produced therefrom can be used in various immunodiagnostic techniques known to those skilled in the art, including ELISA and microarray-related techniques. In addition, these reagents can be used to evaluate antibody responses, including serum antibody levels, against the immunogenic polysaccharide conjugates. The assay methodology of the present invention can involve the use of labels, such as fluorescent, chemiluminescent, radioactive, enzyme-labeled or dye molecules, and / or secondary immunological reagents for direct or indirect detection of the complex between the antigen or antibody in the biological sample and the corresponding antibody or antigen bound to a solid support.

[0313] The antibodies or antibody fragments produced may also be useful in passive immunotherapy or for prophylaxis against streptococcal infections.

[0314] Methods for Producing Polysaccharides In yet another aspect, the invention relates to a method for producing the polysaccharides described herein, comprising culturing GBS and harvesting the polysaccharides produced by the bacteria. In one embodiment, the GBS comprises S. agalactiae. The bacteria may be any strain of S. agalactiae. In a preferred embodiment, the bacteria is an encapsulated strain of S. agalactiae. S. agalactiae strains for use in the present invention include 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524, MB4052 (ATCC Accession No. 31574), H36B (ATCC Accession No. 12401), S40, S42, MB4053 (ATCC Accession No. 31575), M709, 133, 7 357, PFEGBST0267, MB4055 (ATCC accession no. 31576), 18RS21 (ATCC accession no. BAA-1175), S16, S20, V8 (ATCC accession no. 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB4082 (ATCC accession no. 31577), M132, 110, M781 (ATCC accession no. BAA-22), D136C(3)(AT ATCC accession no. 12403), M782, S23, 120, MB4316 (M-732; ATCC accession no. 31475), M132, K79, COH1 (ATCC accession no. BAA-1176), PFEGBST0563, 3139 (ATCC accession no. 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession no. BAA-23), CJB112, and 2603 V / R (ATCC Accession No. BAA-611), NCTC10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B4589, B4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62, and IT-PW-64.

[0315] The polysaccharides described herein can be produced by culturing GBS in a suitable medium. The suitable medium can include Columbia broth. The medium can include dextrose, hemin and / or glucose. Preferably, the medium includes Columbia broth and dextrose. When S. agalactiae is cultured using Columbia broth and dextrose, preferably the temperature for culturing is 20 to 40°C, preferably 37°C. In a preferred embodiment, the bacteria is cultured under aerobic conditions. In another preferred embodiment, the bacteria is cultured for 12 to 60 hours.

[0316] The polysaccharides may be collected from the obtained culture by using methods known in the art for collecting target substances from cultures, such as heating, enzyme treatment, centrifugation, precipitation, treatment with activated charcoal, and / or filtration (see, e.g., U.S. Patent Application Publication Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498; International Patent Application Publication No. WO2008 / 118752). In one embodiment, the culture containing the bacteria and polysaccharides is centrifuged and treated with enzymes, such as lysozyme, RNase, DNase, pronase, mutanolysin, and mixtures thereof. For example, in one embodiment, a suitable organic solvent is added to the obtained supernatant to precipitate proteins, and the precipitate is removed by centrifugation. The polysaccharides may then be precipitated by further adding a suitable organic solvent to the supernatant, and the polysaccharides may be collected by centrifugation. More specifically, the polysaccharides described herein may be obtained by adding ethanol to the bacteria-removed supernatant at a final concentration of about 25% by volume, removing the protein-containing precipitate by centrifugation, further adding ethanol thereto to a final concentration of about 75% by volume, and then collecting the precipitate by centrifugation. The resulting precipitate may be dried with nitrogen. The resulting precipitate may be resuspended in Tris and 0.05% Na azide.

[0317] A further aspect of the present invention provides a novel method using organic reagents such as derivatized hydroxylamine compounds for the isolation of nearly intact high molecular weight CPs while preserving N- and O-acetyl groups. Because this method does not lyse cells, CPs isolated by centrifugation are minimally contaminated by intracellular components, leading to higher overall yields. Moreover, these reagents cleave the B antigen impurity through its multiple phosphodiester bonds into very small fragments that can be easily removed by hemodiafiltration.

[0318] In one embodiment, the CP is isolated by reacting hydroxylamine with a cell paste containing the capsular polysaccharide-producing bacteria. In certain embodiments, the method further comprises a centrifugation step. In other embodiments, the method further comprises a filtering step. In yet other embodiments, the capsular polysaccharide-producing bacteria is selected from the group consisting of Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, and the like. typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium and Enterococcus faecalis.

[0319] In certain aspects of the invention, the hydroxylamine may be one listed in Table 2 of Example 2. In preferred embodiments, the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine, diethylhydroxylamine, hydroxylamine, ethylenediamine, triethylenetetramine, 1,1,4,7,10,10 hexamethyltriethylenetetramine, and 2,6,10,trimethyl 2,6,10 triazaundecane.

[0320] In one embodiment of the invention, the concentration of hydroxylamine is from about 5 mM to about 200 mM, e.g., from about 5 mM to about 150 mM, from about 5 mM to about 100 mM, from about 5 mM to about 75 mM, from about 5 mM to about 50 mM, from about 5 mM to about 25 mM, from about 5 mM to about 10 mM, 10 mM to about 200 mM, e.g., from about 10 mM to about 150 mM, from about 10 mM to about 100 mM, from about 10 mM to about 75 mM, from about 10 mM to about 50 mM, from about 10 mM to about 25 mM. M, about 25 mM to about 200 mM, about 25 mM to about 150 mM, about 25 mM to about 100 mM, about 25 mM to about 75 mM, about 25 mM to about 50 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, 50 mM to about 100 mM, and about 50 mM to about 75 mM, etc.

[0321] In another embodiment, the pH of the reaction is maintained at about 5.5 to about 9.5, e.g., about 5.5 to about 9.0, about 5.5 to about 8.5, about 5.5 to about 8.0, about 5.5 to about 7.5, about 5.5 to about 7.0, about 5.5 to about 6.5, about 6.0 to about 9.5, about 6.0 to about 9.0, about 6.0 to about 8.5, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.0 to about 7.0, about 6.5 to about 9.5, about 6.5 to about 8.5, about 6.5 to about 8.0, about 6.5 to about 7.5, about 7.0 to about 9.5, about 7.0 to about 9.0, 7.0 to about 8.5 and about 7.0 to about 8.0.

[0322] In a further embodiment of the invention, the reaction is carried out at a temperature between about 20° C. and about 85° C., e.g., between about 20° C. and about 80° C., between about 20° C. and about 75° C., between about 20° C. and about 70° C., between about 20° C. and about 65° C., between about 20° C. and about 60° C., between about 20° C. and about 55° C., between about 20° C. and about 50° C., between about 25° C. and about 85° C., between about 25° C. and about 80° C., between about 25° C. and about 75° C., between about 25° C. and about 55° C., between about 25° C. and about 50° C., to about 70°C, about 25°C to about 65°C, about 25°C to about 60°C, about 25°C to about 55°C, about 25°C to about 50°C, about 30°C to about 85°C, about 30°C to about 80°C, about 30°C to about 75°C, about 30°C to about 70°C, about 30°C to about 65°C, about 30°C to about 60°C, about 30°C to about 55°C, about 30°C to about 50°C, about 35°C to about 85°C , about 35°C to about 80°C, about 35°C to about 75°C, about 35°C to about 70°C, about 35°C to about 65°C, about 35°C to about 60°C, about 35°C to about 55°C, about 40°C to about 85°C, about 40°C to about 80°C, about 40°C to about 75°C, about 40°C to about 70°C, about 40°C to about 65°C, about 40°C to about 60°C, about 45°C to about 85°C, about 45°C from about 80°C, from about 45°C to about 75°C, from about 45°C to about 70°C, from about 45°C to about 65°C, from about 50°C to about 85°C, from about 50°C to about 80°C, from about 50°C to about 75°C, from about 50°C to about 70°C, from about 55°C to about 85°C, from about 55°C to about 80°C, from about 55°C to about 75°C, from about 60°C to about 85°C, and from about 65°C to about 85°C.

[0323] In yet another embodiment, the reaction time is from about 10 hours to about 90 hours, e.g., from about 10 hours to about 85 hours, from about 10 hours to about 80 hours, from about 10 hours to about 75 hours, from about 10 hours to about 70 hours, from about 10 hours to about 60 hours, from about 10 hours to about 50 hours, from about 10 hours to about 40 hours, from about 10 hours to about 30 hours, from about 10 hours to about 25 hours, from about 10 hours to about 20 hours, from about 10 hours to about 15 hours, from about 15 hours to about 90 hours, from about 15 hours to about 85 hours, from about 15 hours to about 80 hours, from about 15 hours to about 20 hours, about 75 hours, about 15 hours to about 70 hours, about 15 hours to about 60 hours, about 15 hours to about 50 hours, about 15 hours to about 40 hours, about 15 hours to about 30 hours, 15 hours to about 20 hours, such as about 20 hours to about 90 hours, about 20 hours to about 85 hours, about 20 hours to about 80 hours, about 20 hours to about 75 hours, about 20 hours to about 70 hours, about 20 hours to about 60 hours, about 20 hours to about 50 hours, about 20 hours to about 40 hours, about 20 hours to about 30 hours, and about 20 hours to about 25 hours.

[0324] Alternatively, in another embodiment of the invention, the polysaccharides are chemically synthesized. The polysaccharides may be chemically synthesized according to conventional methods.

[0325] In yet another embodiment of the invention, the polysaccharides are prepared by cloning and expressing a biosynthetic pathway for producing the polysaccharide, followed by expression in a surrogate host. For example, a host cell may be modified to produce a polysaccharide having structural similarity to the polysaccharides described herein, where the repeating units of the polysaccharides produced in the host cell are partially identical to the repeating units of the polysaccharides described herein. The host cell is structurally similar to the polysaccharides described herein, e.g., if the repeating units of the polysaccharide have unknown branching, they are non-uniform in size and / or in branching arrangement when compared to the repeating units of the polysaccharides described herein. Preferably, the host cell is a bacterial host cell. EXAMPLES

[0326] The following examples demonstrate some embodiments of the present invention. However, it should be understood that these examples are for illustration only and do not claim to be completely definitive on the conditions and scope of the present invention. When typical reaction conditions (e.g., temperature, reaction time, etc.) are given, it should be understood that conditions both above and below the specified ranges can also be used, although generally less favorable. All parts and percentages referred to herein are by weight, and all temperatures are expressed in degrees Celsius unless otherwise specified.

[0327] Additionally, the following examples were performed using standard techniques that are well known and routine to those skilled in the art, unless otherwise specifically described. As noted above, the following examples are presented for illustrative purposes and should not be construed as limiting the scope of the invention in any way.

[0328] Example 1 Preparation of polysaccharide-protein conjugates using O-deacetylated polysaccharides S. agalactiae strains for each serotype were fermented in submerged culture with pH control in defined media. The procedures and media used were optimized through experimentation and were an extension of the basic technique previously described in von Hunolstein, C. et al., Appl. Micro. Biotech. 38(4):458-462 (1993). Capsular polysaccharide was removed from the cells by NaOH treatment. After clarification, a series of UF / DF, precipitation and carbon filtration steps yielded the purified polysaccharide. See, e.g., U.S. Pat. No. 8,652,480. The activated polysaccharide was conjugated with CRM197 using reductive amination chemistry. See, e.g., U.S. Pat. No. 5,360,897.

[0329] Example 2 Isolation of O-acetylated polysaccharides Cell paste from GBS capsular polysaccharide (CP) serotype Ia obtained after heat killing and centrifugation of fermentation broth (1.2 L) was resuspended in 175 mL of 25 mM potassium phosphate buffer (25 mM, pH 6.9). The suspension was mixed with aqueous hydroxylamine O-sulfonic acid solution to a final concentration of 10 mM. The pH of the suspension was determined to be about 5.8. The suspension was stirred at 55° C. for 72 hours. The suspension was then centrifuged at around 10,000 rpm and the supernatant was collected. The supernatant containing the crude cleaved CP was analyzed for molecular weight and yield. The remaining portion was subjected to purification by hemodiafiltration using a 30 kDa MWCO membrane using water for injection (WFI). The purified polysaccharide was further analyzed for molecular weight by size exclusion chromatography coupled with a multi-angle light scattering detector (SEC-MALS) (Table 1).

[0330] [Table 1]

[0331] Several hydroxylamine, both nitrogen and oxygen substituted compounds were screened for their activity using the method described above. Yields were calculated by gel permeation chromatography coupled with multi-angle light scattering detection (GPC-MALS) of crude supernatants using refractive index (RI) response and a specific squared refractive index increment (dn / dc) value of 0.135. Yields were determined by optimizing conditions such as type of hydroxylamine, concentration, temperature and reaction time (see Table 2). In general, increasing hydroxylamine concentration, higher temperature and longer reaction time lead to higher yields.

[0332] [Table 2-1]

[0333] [Table 2-2]

[0334] [Table 2-3]

[0335] [Table 2-4]

[0336] Substituted and unsubstituted hydroxylamines were found to be highly effective in releasing GBS capsular polysaccharides from the cell wall. This approach results in the isolation of high molecular weight CPS with preservation of N- and O-acetyl groups. Among several compounds screened, dibenzylhydroxylamine was found to be the most effective. The data are shown in Table 3 ([dibenzylhydroxylamine]- 50 mM; ph- 7-8; temperature- 50 °C; time- 24 h).

[0337] [Table 3]

[0338] Because dibenzylhydroxylamine has poor solubility in water, alternative derivatives of hydroxylamine that are more freely water-soluble than dibenzylhydroxylamine and have similar or higher activity are desirable. After screening a small number of compounds, diethylhydroxylamine was found to be a good alternative. The data are shown in Table 4 ([ diethylhydroxylamine]- 100mM; pH- 7~8; Temperature- 60°C; Time- 19 hours).

[0339] [Table 4]

[0340] Hydroxylamine (NH2-OH) was also found to be effective in cleaving CPS from the cell wall. The data are shown in Table 5 ([hydroxylamine] - 100 mM; pH - 7-7.5; temperature - 65°C; time - 17 hours). For serotype III, the yield was 54% after 17 hours, but the yield increased to a maximum of 70% after 3.5 days.

[0341] [Table 5]

[0342] Screening of Oligoamines for Release of GBS Capsular Polysaccharide from Cells Hydroxylamine and its substituted compounds were found to be very efficient in cleaving capsular polysaccharide from the GBS cell wall. However, they were found to be less efficient for serotypes II and V. Oligoamines were therefore tested in the belief that they might be more active with multiple amine functional groups.

[0343] Ethylenediamine was found to be effective in releasing capsular polysaccharides from all serotypes, and the data are shown in Table 6 ([Ethylenediamine]- 50 or 100 mM; pH 8.0; 16 hours; 80°C; 25 mM EDTA).

[0344] [Table 6]

[0345] Other representative oligoamines were tested for their activity using serotype Ia and V cell pastes, but were also found to be less efficient with serotype V. Data are shown in Tables 7 ([triethylenetetramine]- 100 mM; pH- 8.9; temperature- 60°C; time- 15 hours), 8 ([1,1,4,7,10,10 hexamethyltriethylenetetramine]- 10 mM; pH- 6.3; temperature- 60°C; time- 20 hours), and 9 ([2,6,10, trimethyl 2,6,10 triazaundecane]- 10 mM; pH- 7-8; temperature- 60°C; time- 19 hours).

[0346] [Table 7]

[0347] [Table 8]

[0348] [Table 9]

[0349] Example 3 Activating the conjugated polysaccharide of GBS capsular polysaccharide by reductive amination Polysaccharide oxidation was carried out in 100 mM potassium phosphate buffer (pH 6.0±0.5) by sequential addition of calculated amounts of 500 mM potassium phosphate buffer (pH 6.0) and water for injection (WFI) to obtain a final polysaccharide concentration of 2.0 g / L. If necessary, the reaction pH was adjusted to approximately pH 6.0. After pH adjustment, the reaction temperature was adjusted to 23° C. The oxidation was initiated by the addition of approximately 0.25 molar equivalents of sodium periodate. The oxidation reaction was carried out at 5±3° C. for approximately 16 hours.

[0350] Concentration and hemodiafiltration of the activated polysaccharides were performed using 5K MWCO ultrafiltration cassettes. Hemodiafiltration was performed against 20 diavolumes of WFI. The purified activated polysaccharides were then stored at 5±3° C. The purified activated sugars were characterized by, among others, (i) sugar concentration by colorimetric assay, (ii) aldehyde concentration by colorimetric assay, (iii) degree of oxidation, and (iv) molecular weight by SEC-MALLS.

[0351] The degree of oxidation of the activated polysaccharide (DO = moles of sugar repeating units / moles of aldehyde) is given by It was decided that:

[0352] The number of moles of sugar repeating units is determined by various colorimetric methods, for example, by using the anthrone method. With the anthrone method, polysaccharides are first broken down into monosaccharides by the action of sulfuric acid and heat. The anthrone reagent reacts with hexoses to form a yellow-green complex whose absorbance is read spectrophotometrically at 625 nm. Within the range of the assay, the absorbance is directly proportional to the amount of hexose present.

[0353] The moles of aldehyde are also determined simultaneously using the MBTH colorimetric method. The MBTH assay involves the formation of an azine compound by reacting aldehyde groups (from a given sample) with 3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). Excess 3-methyl-2-benzothiazolone hydrazone is oxidized to form a reactive cation. The reactive cation and azine react to form a blue chromophore. The formed chromophore is then read spectroscopically at 650 nm.

[0354] The activated polysaccharide is combined with sucrose excipient and lyophilized. The activated polysaccharide was blended with sucrose to a ratio of 25 grams of sucrose per gram of activated polysaccharide. The bottles of the blended mixture were then lyophilized. After lyophilization, the bottles containing the lyophilized activated polysaccharide were stored at -20±5°C. The calculated amount of CRM197 protein was shell frozen and lyophilized separately. The lyophilized CRM197 was stored at -20±5°C.

[0355] Reconstitute the lyophilized activated polysaccharide and carrier protein The lyophilized activated polysaccharide was redissolved in anhydrous dimethyl sulfoxide (DMSO). Once the polysaccharide was completely dissolved, an equal volume of anhydrous DMSO was added to the lyophilized CRM197 for redissolution.

[0356] Conjugate and cap The redissolved activated polysaccharide was combined with the redissolved CRM197 in a reaction vessel and then thoroughly mixed to obtain a clear solution before initiating the conjugation with sodium cyanoborohydride. The final polysaccharide concentration in the reaction solution was approximately 1 g / L. The conjugation was initiated by adding 1.0-1.5 MEq of sodium cyanoborohydride to the reaction mixture and incubating at 23 ± 2 °C for 20-48 h. The conjugation reaction was terminated by adding 2 MEq of sodium borohydride (NaBH4) to cap the unreacted aldehydes. The capping reaction was allowed to continue for 3 ± 1 h at 23 ± 2 °C.

[0357] Purifying the conjugate The conjugate solution was diluted 1:10 with chilled 5 mM succinate-0.9% saline (pH 6.0) in preparation for purification by tangential flow filtration using 100-300K MWCO membranes.

[0358] The diluted conjugate solution was passed through a 5 μm filter and hemodiafiltration was performed using 5 mM succinate / 0.9% saline (pH 6.0) as the medium. After hemodiafiltration was completed, the conjugate concentrate was transferred through a 0.22 μm filter. The conjugate was further diluted with 5 mM succinate / 0.9% saline (pH 6) to a target sugar concentration of approximately 0.5 mg / mL. Alternatively, the conjugate was purified by tangential flow filtration using 100-300K MWCO membranes with 20 mM histidine-0.9% saline (pH 6.5). A final 0.22 μm filtration step was completed to obtain the immunogenic conjugate.

[0359] Example 4 Effect of different conjugation conditions on GBS polysaccharide-CRM197 conjugates GBS serotype Ia, Ib, II, III, IV and V conjugates were produced by deliberately varying periodate oxidation / reductive amination chemistry (PO / RAC) conditions, including the solvent for the reagents (aqueous medium vs. DMSO), different levels of sialic acid in the initial polysaccharide, and degree of oxidation / glyco-epitope modification. In general, conjugates produced using DMSO as the solvent were found to have lower levels of unreacted (free) polysaccharide, higher conjugate molecular weight and higher saccharide / protein ratio than conjugates produced using aqueous medium.

[0360] Conjugation processes that produce conjugates with low levels of unreacted (free) polysaccharide are advantageous and preferred, as it is well known that high levels of unreacted (free) polysaccharide can lead to excessive T-cell-independent immune responses that have the potential to dilute the T-cell-dependent response produced by the polysaccharide-protein conjugate, thereby reducing the immunogenic response produced by the conjugate.

[0361] Selected GBS polysaccharides were chemically desialylated by methods known in the art (see Chaffin, DO et al., J Bacteriol 187(13):4615-4626 (2005)) and conjugate variants were produced to determine the effect of % desialylation on immunogenicity. Desialylation above about 40% (i.e., sialic acid levels below about 60%) had a negative effect on immunogenicity.

[0362] Similarly, in most cases, a degree of oxidation below about 5 or more than about 20% glycoepitope modification had a negative effect on immunogenicity. Because oxidation occurs via sialic acid on the capsular polysaccharide, the results seem to indicate that more than about 20% glycoepitope modification reduces the sialic acid content, which leads to reduced immunogenicity.

[0363] Conversely, conjugates with a variety of sugar / protein ratios or polysaccharide molecular weights generated immunogenic responses in mice, indicating a relatively wide range of acceptance criteria for these attributes.

[0364] Additional conjugate variants were also produced using alternative chemical routes. One alternative chemistry involved producing conjugates by reacting polysaccharides with carbonylditriazole (CDT) and carrying out the conjugation reaction in DMSO. In another alternative chemistry, conjugates were produced by oxidation of polysaccharides using TEMPO [(2,2,6,6-tetramethylpiperidin-1-yl)oxyl] reagent (instead of sodium periodate) followed by conjugation using reductive amination chemistry (TEMPO / RAC) in DMSO, as detailed in Example 3 above. All conjugates produced by these alternative chemistries were demonstrated to be immunogenic in mice, indicating the suitability of alternative chemical routes other than PO / RAC. However, some conjugation chemistries performed better with some serotypes than others.

[0365] OPA was performed as in Nanra, JS et al., Hum. Vaccin. Immunother., 9(3):480-487 (2013), substituting group B Streptococcus isolates for Staphylococcus aureus isolates and omitting the pre-opsonization step. Post-dose 3 (PD3) OPA titers are provided as the geometric mean from groups of 10-20 mice immunized with 1 mcg / ml at each dose of the respective conjugate.

[0366] GBS serotype Ia polysaccharide-CRM197 conjugate Conjugates produced using PO / RAC and an activated polysaccharide with a DO of 16-17 (approximately 6% glycoepitope modification) were demonstrated to be immunogenic (conjugates 1 and 3). However, using an activated polysaccharide with a DO of 5.4 (approximately 19% glycoepitope modification) had a negative effect on immunogenicity (conjugate 2). Similarly, a sialic acid level of 50% produced little to no immunogenic response (conjugate 4). The results are shown in Table 10.

[0367] [Table 10]

[0368] Additional conjugate variants were produced using alternative conjugation chemistries and conjugate molecule attributes (results are shown in Table 11). Conjugate 5 was produced by reacting the polysaccharide with carbonylditriazole (CDT), and the conjugation reaction was carried out in DMSO. Conjugate 6 was produced by oxidation of the polysaccharide using TEMPO reagent (instead of sodium periodate), followed by conjugation using reductive amination chemistry in DMSO, as detailed in Example 3 above. Conjugate 7 was produced by deliberately varying the PO / RAC and conjugation parameters to generate a conjugate with a high sugar / protein ratio (SPR). Conjugate 8 was produced by PO / RAC using a polysaccharide with a low MW (40 kDa). All these conjugates were demonstrated to be immunogenic in mice, demonstrating the suitability of alternative conjugation chemistries other than periodate oxidation / reductive amination chemistry, as well as alternative conjugate attributes such as SPR and low MW of the nascent polysaccharide.

[0369] [Table 11]

[0370] GBS serotype Ib polysaccharide-CRM197 conjugate Conjugates produced using PO / RAC and an activated polysaccharide with a DO of 15.8 (approximately 6% sugar epitope modification) in DMSO were demonstrated to be immunogenic in mice (conjugates 9 and 11). Conjugates produced with PO / RAC in DMSO were slightly more immunogenic than those produced with PO / RAC in aqueous media when all other conjugate molecular attributes were the same (conjugates 9 and 11, respectively). However, using an activated polysaccharide with a DO of 4.7 (approximately 21% sugar epitope modification) had a negative effect on immunogenicity (conjugate 10). Immunogenicity was almost completely abolished, with very few responders in the conjugate produced using PO / RAC and 95% desialylated (5% sialic acid level) polysaccharide (conjugate 12). The results are shown in Table 12.

[0371] [Table 12]

[0372] Additional conjugate variants were produced using alternative conjugation chemistries and conjugate molecule attributes (results are shown in Table 13). Conjugate 13 was produced by PO / RAC using a polysaccharide with low sialylation (65%) in the initial polysaccharide. Conjugate 14 was produced by deliberately varying the PO / RAC and conjugation parameters to generate a conjugate with a high sugar / protein ratio (SPR). Conjugate 15 was produced by reacting the polysaccharide with carbonylditriazole (CDT), with the conjugation reaction carried out in DMSO. Conjugate 16 was produced by oxidation of the polysaccharide using TEMPO reagent (instead of sodium periodate), followed by conjugation using reductive amination chemistry in DMSO, as detailed in Example 3 above. All these conjugates were demonstrated to be immunogenic in mice, demonstrating the suitability of alternative conjugation chemistries other than periodate oxidation / reductive amination chemistry, as well as alternative conjugate attributes such as SPR and low MW of the nascent polysaccharide.

[0373] [Table 13]

[0374] GBS serotype II polysaccharide-CRM197 conjugate Conjugates produced using PO / RAC and activated polysaccharides with DOs of 4-15 (approximately 7-23% glycoepitope modification) were demonstrated to be immunogenic in mice (conjugates 17-20). A conjugate produced using PO / RAC and a polysaccharide with a sialylation level of 74% (26% desialylation) was also demonstrated to be immunogenic (conjugate 20). The results are shown in Table 14.

[0375] [Table 14]

[0376] Additional conjugate variants were produced using alternative conjugation chemistries and conjugate molecule attributes (results are shown in Table 15). Conjugates 21 and 22 were produced by deliberately varying the PO / RAC and conjugation parameters to generate conjugates with low and high sugar / protein ratios (SPR), respectively. Conjugate 23 was produced by oxidation of the polysaccharide using TEMPO reagent (instead of sodium periodate), followed by conjugation using reductive amination chemistry in DMSO, as detailed in Example 3 above. Conjugate 24 was produced by reacting the polysaccharide with carbonylditriazole (CDT), and the conjugation reaction was performed in DMSO. All these conjugates were demonstrated to be immunogenic in mice, demonstrating the suitability of alternative conjugation chemistries other than periodate oxidation / reductive amination chemistry, and alternative conjugate attributes such as SPR.

[0377] [Table 15]

[0378] GBS serotype III polysaccharide-CRM197 conjugate Conjugates produced using PO / RAC and activated polysaccharide with a DO of 10-17 (approximately 6-10% glycoepitope modification) in DMSO were demonstrated to be immunogenic in mice (conjugates 25 and 30). Conjugates with a DO of 2.9 (approximately 34% glycoepitope modification) or a high sugar / protein ratio (2.1) (conjugates 26 and 27, respectively) were demonstrated to be relatively poorly immunogenic. A conjugate produced using PO / RAC and a polysaccharide with a sialylation level of 81% (19% desialylated) was demonstrated to be immunogenic (conjugate 30). However, a conjugate produced using a polysaccharide with a sialylation level of 58% (42% desialylated) was demonstrated to be poorly immunogenic (conjugate 29). Conjugates produced by PO / RAC in DMSO were slightly more immunogenic than those produced by PO / RAC in aqueous media (conjugates 25 and 28, respectively) when all other conjugate molecular attributes were equal. The results are shown in Table 16.

[0379] [Table 16]

[0380] Additional conjugate variants were produced using alternative conjugation chemistries and conjugate molecule attributes (results are shown in Table 17). Conjugates 31-35 were produced by deliberately varying the PO / RAC and conjugation parameters to generate conjugates with different MW. Conjugate 36 was produced by reacting the polysaccharide with carbonylditriazole (CDT), and the conjugation reaction was carried out in DMSO. Conjugate 37 was produced by oxidation of the polysaccharide using TEMPO reagent (instead of sodium periodate), followed by conjugation using reductive amination chemistry in DMSO, as detailed in Example 3 above. All these conjugates were demonstrated to be immunogenic in mice, demonstrating the suitability of alternative conjugation chemistries other than periodate oxidation / reductive amination chemistry, and alternative conjugate attributes such as MW. The conjugate produced at a low DO of 5 (approximately 20% glycoepitope modification) was still immunogenic in mice (conjugate 32 in Table 17) compared to the conjugate produced at a DO of 2.9 (approximately 34% glycoepitope modification) (conjugate 26 in Table 16 above).

[0381] [Table 17]

[0382] GBS serotype IV polysaccharide-CRM197 conjugate Conjugates produced using PO / RAC and activated polysaccharides with DOs of 6.9-14.2 (approximately 7-14% glycoepitope modification) were demonstrated to be immunogenic in mice (conjugates 38-41). A conjugate produced using PO / RAC and a polysaccharide with a sialylation level of 60% (40% desialylation) was also demonstrated to be immunogenic (conjugate 41). The results are shown in Table 18.

[0383] [Table 18]

[0384] Additional conjugate variants were produced using alternative conjugation chemistries and conjugate molecular attributes. Results are shown in Table 19. Conjugates 42 and 45 were produced by deliberately varying the PO / RAC and conjugation parameters to generate conjugates with high DO (lower oxidation levels) and high SPR, respectively. Conjugate 43 was produced by reacting the polysaccharide with carbonylditriazole (CDT), and the conjugation reaction was carried out in DMSO. Conjugate 44 was produced by oxidation of the polysaccharide using TEMPO reagent (instead of sodium periodate), followed by conjugation using reductive amination chemistry in DMSO, as detailed in Example 3 above. All serotype IV conjugates were demonstrated to be immunogenic in mice, demonstrating the suitability of alternative conjugation chemistries other than periodate oxidation / reductive amination chemistry, and conjugate attributes such as SPR. Conjugates produced with a DO (lower oxidation) up to at least 20 (approximately 5% glycoepitope modification) were still immunogenic in mice.

[0385] [Table 19]

[0386] GBS serotype V polysaccharide-CRM197 conjugate Conjugates produced using PO / RAC and activated polysaccharides with DOs between 4.4 and 14.6 (approximately 7-23% glycoepitope modification) were demonstrated to be immunogenic in mice (conjugates 46 and 47). The desialylated (5% sialylation level) conjugate was not immunogenic (conjugate 49), and the conjugate produced using the PO / RAC process using an aqueous solvent generated a low immune response (conjugate 48). The results are shown in Table 20.

[0387] [Table 20]

[0388] Additional conjugate variants were produced using alternative conjugation chemistries and conjugate molecule attributes. Results are shown in Table 21. Conjugates 50 and 53 were produced by deliberately varying the PO / RAC and conjugation parameters to generate conjugates with lower sialylation levels (81% sialylation) and lower MW, respectively. Conjugate 51 was produced by reacting the polysaccharide with carbonylditriazole (CDT), and the conjugation reaction was carried out in DMSO. Conjugate 52 was produced by oxidation of the polysaccharide using TEMPO reagent (instead of sodium periodate), followed by conjugation using reductive amination chemistry in DMSO, as detailed in Example 3 above. All serotype V conjugates, except for those produced using CDT chemistry, were demonstrated to be immunogenic in mice, demonstrating the suitability of alternative conjugation chemistries other than periodate oxidation / reductive amination chemistry, and conjugate attributes such as MW. Conjugates produced using CDT chemistry were shown to be significantly less immunogenic compared to other conjugates produced by RAC. The conjugate with 81% sialylation (conjugate 50) provided a lower immune response compared to the conjugate with over 95% sialylation (conjugate 53) but a higher immune response than the conjugate with 5% sialylation (conjugate 49 in Table 20 above).

[0389] [Table 21]

[0390] Example 5 GBS III-CRM197 and GBS V-CRM197 monovalent conjugate vaccines generated OPA responses in mice Female CD-1 mice were subcutaneously immunized three times at weeks 0, 3 and 6 with 1mcg, 0.1mcg or 0.01mcg of Group B Streptococcus (GBS) serotype III conjugated to CRM197 (GBS III-CRM197) or GBS serotype V conjugated to CRM197 (GBS V-CRM197). Sera after dose 3 (PD3) were evaluated by opsonophagocytosis assay (OPA). OPA was performed as described in Example 4. Both conjugates induced OPA responses in mice (Table 22). Samples without detectable OPA responses were assigned a value of 50.

[0391] [Table 22]

[0392] Example 6 GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197 monovalent conjugate vaccines generated OPA responses in mice Six sets of female CD-1 mice were subcutaneously immunized three times at weeks 0, 3, and 6 with a vaccine containing 1 mcg of each GBS capsular polysaccharide (CP) conjugated to CRM197. Initial studies showed that the mice had no pre-existing OPA titers to any of the six serotypes tested. Sera from PD3 were analyzed by OPA against the cognate GBS serotype contained in the vaccine. OPA was performed as described in Example 4. The results are shown in Tables 23 and 24 below.

[0393] [Table 23]

[0394] [Table 24]

[0395] Example 7 Opsonic activity of sera compared with IgG isolated from mice immunized with GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197, and GBS V-CRM197 monovalent conjugate vaccines Female CD-1 or BALB / c mice were immunized subcutaneously three times at weeks 0, 3, and 6 with individual GBS CPs conjugated to 1 mcg of CRM197. Either AlPO4 or QS-21 was used as adjuvant. PD3 serum was tested with serotype-specific OPAs, and immunoglobulin G fractions were then isolated and tested for OPA activity. Purified IgG OPA activity was normalized to 5 mg / ml (within the range of IgG amounts in normal mouse serum). All six GBS CPS conjugates elicited IgG antibodies with opsonic activity (Figure 1).

[0396] Example 8 GBS Ia-TT, GBS Ib-TT, GBS II-TT, GBS III-TT, GBS IV-TT and GBS V-TT, monovalent conjugate vaccines generated OPA responses in rabbits Rabbits were immunized three times with GBS serotype Ia polysaccharide conjugated to tetanus toxoid at 50 mcg / ml, GBS serotype Ib polysaccharide conjugated to tetanus toxoid at 10 mcg / ml, GBS serotype II polysaccharide conjugated to tetanus toxoid at 50 mcg / ml, GBS serotype III polysaccharide conjugated to tetanus toxoid at 50 mcg / ml, GBS serotype IV polysaccharide conjugated to tetanus toxoid at 50 mcg / ml, or GBS serotype V polysaccharide conjugated to tetanus toxoid at 50 mcg / ml, adjuvanted with complete Freund's adjuvant for the first dose and incomplete Freund's adjuvant for the second and third doses. Conjugates were produced using polysaccharides with sialic acid levels greater than 95% and CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) chemistry. PD3 immune responses were measured by OPA as described in Example 4. Serum titers are shown in Table 25, while purified IgG titers are shown in Table 26 below. GBS serotype Ia, Ib, II, III, IV and V polysaccharides conjugated with TT were highly immunogenic in rabbits.

[0397] [Table 25]

[0398] [Table 26]

[0399] Example 9 Hexavalent GBS conjugate vaccine generated OPA responses in non-human primates Three groups of rhesus macaques were immunized intramuscularly three times at weeks 0, 4 and 8 with a hexavalent group B streptococcus (GBS6) vaccine. The GBS6 vaccine included GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197. Two groups included aluminum phosphate (AlPO4) as an adjuvant and were dosed with either 5mcg of each conjugate or 50mcg of each conjugate. The third group was dosed with 5mcg of each conjugate and did not contain an adjuvant. Table 27 below describes the immunization schedule.

[0400] [Table 27]

[0401] Pre-immune sera and sera from PD3 were analyzed by OPA for all six GBS serotypes contained in the vaccine. OPA was performed as described in Example 4. The results are shown in Tables 28 and 29 below. For all six serotypes, the AlPO4-adjuvanted formulations elicited detectable OPA responses (increase in titers from before PD3 or >1 fold increase pre / PD3). The unadjuvanted 5mcg / conjugate dose elicited detectable OPA responses for five of the six serotypes.

[0402] [Table 28]

[0403] [Table 29]

[0404] Example 10 Hexavalent GBS conjugate vaccine generated OPA responses in rats Female Sprague-Dawley rats were immunized subcutaneously twice with 5 mcg / ml of each conjugate in the GBS6 polysaccharide conjugate vaccine as described in Example 9 formulated either with or without aluminum phosphate (AlPO4). Pre-immunization (baseline) and post-dose 2 sera were assessed for OPA assay titers against all six cognate GBS serotypes. OPA titers were measured for each serotype in a GBS384-well assay format and fold increases were calculated. Rats administered the GBS6 vaccine had robust functional antibody responses against each serotype after the second dose, with 7- to 205-fold increases between serotypes in the absence of AlPO4, whereas in its presence this ranged from 11- to 294-fold (Table 30).

[0405] [Table 30]

[0406] Example 11 Immunization of pregnant dams with monovalent or hexavalent GBS glycoconjugate vaccines protects their offspring from GBS III or V infection after birth Female CD-1 mice were administered the GBS6 vaccine, GBS6 vaccine as described in Example 9, containing 5 mcg / ml of each conjugate and 100 mcg / ml of AlPO4. Mice were immunized subcutaneously three times with either III or V monovalent glycoconjugate vaccines (containing 10 mcg / ml of conjugate and 100 mcg / ml of AlPO4, respectively), or vehicle control alone. Mice were allowed to give birth before the third immunization. Pups of immunized mice were inoculated with a lethal dose of either GBS serotype III or GBS serotype V bacteria according to the vaccine received, and survival was monitored over 90 hours. Dam immunization with GBS6+AlPO4 or GBS III-CRM197+AlPO4 provided significant protection (p<0.0001) to the offspring against lethal GBS serotype III challenge. Similarly, dam immunization with GBS V-CRM197+AlPO4 provided significant protection (p<0.0001) to the offspring against lethal GBS serotype V challenge. Results are shown in Table 31.

[0407] [Table 31]

[0408] Example 12 Passive immunization of infant rats with GBS III monoclonal antibodies was protective. Group B Streptococcus serotype III (GBS III) monoclonal antibodies (mAbs) were produced by immunizing mice with a pentavalent vaccine containing serotypes Ia, Ib, II, III, and V. GBS III-specific mAb clones were selected and mAbs recognizing the CP of GBS III were produced using standard procedures. GBS serotype III mAbs were administered passively to suckling rats (n=10 per group; two independent experiments shown) 16 hours prior to inoculation with a clinical GBS III isolate. Four hours after inoculation, blood was collected and remaining CFUs were counted. Treatment with GBS III mAbs reduced the recovered CFUs in suckling rats by more than 4 logs (Table 32).

[0409] [Table 32]

[0410] Example 13 Passive immunization of pregnant mice with GBS Ib, III, and V monoclonal antibodies provided protection in their offspring after birth. Monoclonal antibodies (mAbs) were produced using standard procedures from mice immunized with the pentavalent vaccine (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197 and GBS V-CRM197). mAbs were then identified that specifically recognized the capsular polysaccharides of each of the five serotypes. A 500 mcg / ml dose of GBS serotype Ib (GBS Ib) mAb, GBS serotype III (GBS III) mAb, GBS serotype V (GBS V) mAb or an isotype-matched control mAb was passively administered to pregnant mice approximately 24-48 hours prior to birth. 24-48 hours after birth, pups of immunized murine dams were inoculated with a lethal dose of GBS Ib, GBS III or GBS V bacteria. Survival was monitored over a 96-hour period. Significantly greater survival was observed in pups born to dams immunized with GBS Ib mAb, GBS III mAb, or GBS V mAb compared with control mAb following GBS challenge (Table 33).

[0411] [Table 33]

[0412] Example 14 Stability of GBS conjugates GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197 were independently formulated in 10 mM succinate-phosphate and 155 mM NaCl at various pH levels to test the stability of the conjugates at accelerated storage conditions. The percent change in molecular weight as determined by SEC MALLS was measured after 4 weeks of storage at 50° C. The results are shown in Figures 2-7.

[0413] GBS Ia-CRM197, GBS Ib-CRM197, GBS III-CRM197 and GBS IV-CRM197 conjugates were tested for sialylation stability under various buffer conditions shown in Table 34. Free sialic acid (N-acetylneuraminic acid; NANA) was measured using HPLC after 1 month of storage at 37° C. The results are shown in FIG.

[0414] Both studies suggested that the conjugates performed well at a pH above 6.0, optimally at about pH 6.5.

[0415] [Table 34]

[0416] Example 15 GBS6 preparation To determine the choice of buffer, GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197 and GBS V-CRM197 (GBS6) were formulated together using the same buffer conditions as shown in Table 33 above. The actual pH of the formulations was tested at the following time points: 0 (when the formulations were made), after 1 month at 5° C., after 1 month at 25° C., and after 1 month at 37° C. A shift in pH was observed in the formulations buffered with succinate, whereas no shift was observed in the formulations buffered with histidine. The results are shown in Figures 9-10.

[0417] The effect of histidine buffer concentration on aluminum binding of GBS conjugates was also examined. Formulations containing 150 mM NaCl, 0.01% polysorbate-80, and 0.5 mg / ml aluminum as AlPO4 at pH 6.5 were tested with two different concentrations of conjugate (10 mcg / ml and 40 mg / ml of each serotype) and several different concentrations of histidine. The percent of conjugate bound to aluminum was determined by measuring the total amount of each of the conjugates in the vaccine and the amount of each of the conjugates bound to aluminum. Bound conjugate was measured by centrifuging the vaccine formulation, resuspending the aluminum pellet, solubilizing the aluminum, and measuring bound conjugate using a turbidimetric method using serotype-specific polyclonal antibodies against each of the serotypes. The results are shown in Figures 11-12. It was found that the concentration of histidine buffer affected the percent of each serotype bound to aluminum, with the effect being more pronounced at the lower doses than at the higher doses.

[0418] Agitation studies were performed to determine the amount of polysorbate-80 (PS80) that would be desirable. GBS6 formulations containing 20 mM histidine, 150 mM NaCl, 0.5 mg / ml AlPO4 (if present) and either no PS80, 0.01% PS80, 0.02% PS80 or 0.03% PS80 at pH 6.5 were tested for percentage of total antigenicity loss upon agitation stress. Syringes pre-filled with the formulations were agitated at 500 RPM for 72 hours at room temperature. Control samples (not agitated) were stored at room temperature for 72 hours. The results are shown in Figure 13.

[0419] The concentration of aluminum in the GBS6 formulation was also studied to determine the effect on GBS conjugate binding to aluminum. GBS6 formulations containing 10 mM histidine, 150 mM NaCl, 0.02% PS80, and either 0.25 mg / ml, 0.5 mg / ml, 0.75 mg / ml, or 1.0 mg / ml aluminum as aluminum phosphate (AlPO4) at pH 6.5 were tested for the percent of conjugate bound to aluminum. The percentage of binding to AlPO4 increases as the AlPO4 concentration increases. The results are shown in Figure 14.

[0420] Example 16 GBS6 lyophilized preparation Various lyophilized formulations of GBS6 (GBS Ia-CRM197, GBS Ib-CRM197, GBS II-CRM197, GBS III-CRM197, GBS IV-CRM197, and GBS V-CRM197) were tested for stability. Low (10 mcg / ml) and high (50 mcg / ml) dose formulations were lyophilized containing 20 mM histidine at pH 6.5, 0.02% PS80, approximately 28 mM NaCl, and either 5.5%, 7.0% or 8.5% (w / v) sucrose. The stability of the lyophilized formulations was tested by measuring pH and moisture after 4 months at 5° C., 4 months at 37° C., and 1 month at 50° C. All formulations were stable based on pH and moisture (data not shown). In addition, the percentage of antigenic recovery for each serotype was tested for all formulations after 1, 4 and 9 months at both 5° C. and 37° C., and after 1, 2 and 4 weeks at 50° C. The results are shown in Figures 15-20.

[0421] The following variations in excipients were also prepared and evaluated for the 40 mcg / ml dose of GBS6 formulation: 1) 7% (w / v) sucrose, 2) 2% (w / v) sucrose and 4% (w / v) mannitol, 3) 3% (w / v) sucrose and 3% (w / v) mannitol, 4) 2% (w / v) sucrose and 4% (w / v) glycine, or 5) 3% (w / v) sucrose and 3% (w / v) glycine. All five formulations were pH and moisture stable after 3 months at 5°C, 3 months at 25°C, 3 months at 37°C, and 1 month at 50°C (data not shown). In addition, the percentage of antigenic recovery for each serotype was tested for all formulations after 1, 3, and 7 months at 52-8°C, 25°C, and 37°C, and after 1, 2, and 4 weeks at 50°C. The results are shown in Figures 21 to 25.

[0422] The percent of antigen bound to the aluminum phosphate adjuvant was tested for GBS6 vaccine in reconstituted lyophilized and liquid formulations using turbidimetric methods. Both lyophilized and liquid formulations were prepared at low (10 mcg / ml) and high (50 mcg / ml) doses containing 20 mM histidine, 0.02% PS80, 7.0% (w / v) sucrose, and 500 mcg / ml aluminum as aluminum phosphate. Various concentrations of sodium chloride (NaCl) were also tested to determine the effect on antigen binding. The results for the lyophilized and liquid formulations are shown in Tables 35 and 36, respectively. The low dose formulations were formulated with NaCl concentrations of about 150 mM or greater for both the lyophilized and liquid compositions. had comparable results when used.

[0423] [Table 35]

[0424] [Table 36]

[0425] Aspects of the invention The following clauses describe additional embodiments of the present invention:

[0426] C1. An immunogenic polysaccharide-protein conjugate comprising a Group B Streptococcus (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0427] C2. An immunogenic conjugate of C1, wherein the capsular polysaccharide is selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

[0428] C3. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype Ia.

[0429] C4. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype Ib.

[0430] C5. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype II.

[0431] C6. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype III.

[0432] C7. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype IV.

[0433] C8. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype V.

[0434] C9. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype VI.

[0435] C10. An immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype VII.

[0436] C11. An immunogenic conjugate of C2, in which the capsular polysaccharide is serotype VIII.

[0437] C12. An immunogenic conjugate of C2, in which the capsular polysaccharide is serotype IX.

[0438] C13. The immunogenic conjugate of any one of C1-C12, wherein the capsular polysaccharide has a sialic acid level of greater than about 95%.

[0439] C14. The immunogenic conjugate of any one of C1-C13, wherein the capsular polysaccharide has a sialic acid level of about 100%.

[0440] C15. The immunogenic conjugate of any one of C1-C14, wherein the capsular polysaccharide has at least about 0.6 mM sialic acid per mM polysaccharide.

[0441] C16. The immunogenic conjugate of any one of C1-C15, wherein the capsular polysaccharide has at least about 0.65 mM sialic acid per mM polysaccharide.

[0442] C17. The immunogenic conjugate of any one of C1-C16, wherein the capsular polysaccharide has at least about 0.7 mM sialic acid per mM polysaccharide.

[0443] C18. The immunogenic conjugate of any one of C1-C17, wherein the capsular polysaccharide has at least about 0.75 mM sialic acid per mM polysaccharide.

[0444] C19. The immunogenic conjugate of any one of C1-C18, wherein the capsular polysaccharide has at least about 0.8 mM sialic acid per mM polysaccharide.

[0445] C20. The immunogenic conjugate of any one of C1-C19, wherein the capsular polysaccharide has at least about 0.85 mM sialic acid per mM polysaccharide.

[0446] C21. The immunogenic conjugate of any one of C1-C20, wherein the capsular polysaccharide has at least about 0.9 mM sialic acid per mM polysaccharide.

[0447] C22. The immunogenic conjugate of any one of C1-C21, wherein the capsular polysaccharide has at least about 0.95 mM sialic acid per mM polysaccharide.

[0448] C23. The immunogenic conjugate of any one of C1-C22, wherein the capsular polysaccharide has a molecular weight between about 5 kDa and about 1,000 kDa.

[0449] C24. The immunogenic conjugate of any one of C1-C23, wherein the capsular polysaccharide has a molecular weight of between about 25 kDa and about 750 kDa.

[0450] C25. The immunogenic conjugate of any one of C1-C24, wherein the capsular polysaccharide has a molecular weight of between about 25 kDa and about 400 kDa.

[0451] C26. C1, wherein the capsular polysaccharide has a molecular weight between about 25 kDa and about 200 kDa. The immunogenic conjugate of any one of claims 1 to 25.

[0452] C27. The immunogenic conjugate of any one of C1-C26, wherein the capsular polysaccharide has a molecular weight of between about 100 kDa and about 400 kDa.

[0453] C28. The immunogenic conjugate of any one of C1-27, wherein the molecular weight of the conjugate is between about 300 kDa and about 20,000 kDa.

[0454] C29. The immunogenic conjugate of any one of C1-C28, wherein the molecular weight of the conjugate is between about 1,000 kDa and about 15,000 kDa.

[0455] C30. The immunogenic conjugate of any one of C1-C29, wherein the molecular weight of the conjugate is between about 1,000 kDa and about 10,000 kDa.

[0456] C31. The immunogenic conjugate of any one of C1-C30, wherein the capsular polysaccharide is between about 0% and about 40% O-acetylated.

[0457] C32. The immunogenic conjugate of any one of C1-C31, wherein the capsular polysaccharide is less than about 5% O-acetylated.

[0458] C33. The immunogenic conjugate of any one of C1-C32, wherein the capsular polysaccharide is less than about 4% O-acetylated.

[0459] C34. The immunogenic conjugate of any one of C1-C33, wherein the capsular polysaccharide is less than about 3% O-acetylated.

[0460] C35. The immunogenic conjugate of any one of C1-C34, wherein the capsular polysaccharide is less than about 2% O-acetylated.

[0461] C36. The immunogenic conjugate of any one of C1-C35, wherein the capsular polysaccharide is less than about 1% O-acetylated.

[0462] C37. The immunogenic conjugate of any one of C1-C36, wherein the capsular polysaccharide comprises at least about 0.1 mM O-acetate per mM saccharide repeat unit.

[0463] C38. The immunogenic conjugate of any one of C1-C37, wherein the capsular polysaccharide comprises at least about 0.2 mM O-acetate per mM saccharide repeat unit.

[0464] C39. The immunogenic conjugate of any one of C1-C38, wherein the capsular polysaccharide comprises at least about 0.3 mM O-acetate per mM saccharide repeat unit.

[0465] C40. The immunogenic conjugate of any one of C1-C39, wherein the capsular polysaccharide comprises at least about 0.35 mM O-acetate per mM saccharide repeat unit.

[0466] C41. The immunogenic conjugate of any one of C1-C40, wherein the capsular polysaccharide comprises at least about 0.4 mM O-acetate per mM saccharide repeat unit.

[0467] C42. The immunogenic conjugate of any one of C1-C41, wherein the capsular polysaccharide comprises less than about 0.01 mM O-acetate per mM saccharide repeat unit.

[0468] C43. The immunogenic conjugate of any one of C1-C42, wherein the capsular polysaccharide comprises less than about 0.05 mM O-acetate per mM saccharide repeat unit.

[0469] C44. The immunogenic conjugate of any one of C1-C43, wherein the capsular polysaccharide comprises less than about 0.04 mM O-acetate per mM saccharide repeat unit.

[0470] C45. The immunogenic conjugate of any one of C1-C44, wherein the capsular polysaccharide comprises less than about 0.03 mM O-acetate per mM saccharide repeat unit.

[0471] C46. The immunogenic conjugate of any one of C1-C45, wherein the capsular polysaccharide comprises less than about 0.02 mM O-acetate per mM saccharide repeat unit.

[0472] C47. The immunogenic conjugate of any one of C1-C46, wherein each polysaccharide is independently conjugated to a carrier protein.

[0473] C48. The immunogenic conjugate of any one of C1-C47, wherein the carrier protein is CRM197 or tetanus toxoid.

[0474] C49. The immunogenic conjugate of any one of C1-C48, wherein the carrier protein is CRM197.

[0475] C50. A method for isolating capsular polysaccharide, comprising reacting an organic reagent with a cell broth containing a capsular polysaccharide producing bacterium.

[0476] C51. A method of C50 that does not lyse bacteria.

[0477] C52. Killing bacteria by heat, method C50 or C51.

[0478] C53. The method of any one of C50-C52, further comprising the step of centrifugation to provide a cell paste.

[0479] C54. The method of any one of C50 to C53, further comprising a filtering step.

[0480] C55. The method of C54, wherein said filtering step is hemodiafiltration.

[0481] C56. Capsular polysaccharide-producing bacteria are Streptococcus agalactiae, Streptococcus pneumoniae, 20. The method of any one of C50 to C55, wherein the bacterial strain selected from the group consisting of: Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium and Enterococcus faecalis.

[0482] C57. The method of C56, wherein the bacterium is Streptococcus agalactiae.

[0483] C58. The method of any one of C50 to C57, wherein the organic reagent is a derivatized hydroxylamine compound.

[0484] C59. The method of any one of C50-58, wherein the hydroxylamine is any hydroxylamine listed in Table 2 of Example 2.

[0485] C60. The process of any one of C50 to C59, wherein the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine, diethylhydroxylamine, hydroxylamine, ethylenediamine, triethylenetetramine, 1,1,4,7,10,10 hexamethyltriethylenetetramine, and 2,6,10, trimethyl 2,6,10 triazaundecane.

[0486] C61. The method of any one of C50-C60, wherein the concentration of hydroxylamine is from about 5 mM to about 200 mM.

[0487] C62. The method of any one of C50-C61, wherein the pH of the reaction is from about 5.5 to about 9.5.

[0488] C63. The process of any one of C50-C62, wherein the reaction occurs at a temperature of about 20° C. to about 85° C.

[0489] C64. The process of any one of C50 to C63, wherein the reaction time is from about 10 hours to about 90 hours.

[0490] C65. A method of making an immunogenic polysaccharide-protein conjugate of any one of C1 to C49, wherein the capsular polysaccharide is isolated according to the method of any one of C50 to C64.

[0491] C66. An immunogenic polysaccharide-protein conjugate comprising a capsular polysaccharide prepared by the method of any one of C50 to C64.

[0492] C67. An immunogenic composition comprising the immunogenic polysaccharide-protein conjugate of any one of C1-C49 or C66.

[0493] C68. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharide from Group B Streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

[0494] C69. The immunogenic composition of C68, wherein at least one additional serotype is Ia.

[0495] C70. An immunogenic composition of C69, further comprising a conjugate comprising capsular polysaccharide from GBS serotype Ib.

[0496] C71. An immunogenic composition of C69 or C70, further comprising a conjugate comprising capsular polysaccharide from GBS serotype II.

[0497] C72. The immunogenic composition of any one of C69-C71, further comprising a conjugate comprising capsular polysaccharide from GBS serotype III.

[0498] C73. The immunogenic composition of any one of C69 to C72, further comprising a conjugate comprising capsular polysaccharide from GBS serotype V.

[0499] C74. The immunogenic composition of any one of C69-C73, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VI.

[0500] C75. The immunogenic composition of any one of C69-74, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0501] C76. The immunogenic composition of any one of C69-C75, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0502] C77. The immunogenic composition of any one of C69 to C76, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0503] C78. The immunogenic composition of C68, wherein at least one additional serotype is Ib.

[0504] C79. The immunogenic composition of C78, ​​further comprising a conjugate comprising capsular polysaccharide from GBS serotype II.

[0505] C80. The immunogenic composition of C78 or C79, further comprising a conjugate comprising capsular polysaccharide from GBS serotype III.

[0506] C81. The immunogenic composition of any one of C78-C80, further comprising a conjugate comprising capsular polysaccharide from GBS serotype V.

[0507] C82. The immunogenic composition of any one of C78-C81, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VI.

[0508] C83. The immunogenic composition of any one of C78-C82, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0509] C84. The immunogenic composition of any one of C78-C83, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0510] C85. The immunogenic composition of any one of C78-C84, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0511] C86. The immunogenic composition of C68, wherein at least one additional serotype is II.

[0512] C87. The immunogenic composition of C86, further comprising a conjugate comprising capsular polysaccharide from GBS serotype III.

[0513] C88. The immunogenic composition of C86 or C87, further comprising a conjugate comprising capsular polysaccharide from GBS serotype V.

[0514] C89. The immunogenic composition of any one of C86-C88, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VI.

[0515] C90. The immunogenic composition of any one of C86-C89, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0516] C91. The immunogenic composition of any one of C86 to C90, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0517] C92. The immunogenic composition of any one of C86-C91, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0518] C93. The immunogenic composition of C68, wherein at least one additional serotype is III.

[0519] C94. The immunogenic composition of C93, further comprising a conjugate comprising capsular polysaccharide from GBS serotype V.

[0520] C95. The immunogenic composition of C93 or C94, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VI.

[0521] C96. The immunogenic composition of any one of C93-C95, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0522] C97. The immunogenic composition of any one of C93-C96, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0523] C98. The immunogenic composition of any one of C93-C97, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0524] C99. The immunogenic composition of C68, wherein at least one additional serotype is V.

[0525] C100. An immunogenic composition of C99, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VI.

[0526] C101. The immunogenic composition of C99 or C100, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0527] C102. The immunogenic composition of any one of C99-C101, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0528] C103. The immunogenic composition of any one of C99-C102, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0529] C104. The immunogenic composition of C68, wherein at least one additional serotype is VI.

[0530] C105. The immunogenic composition of C104, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0531] C106. The immunogenic composition of C104 or C105, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0532] C107. The immunogenic composition of any one of C104-C106, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0533] C108. The immunogenic composition of C68, wherein at least one additional serotype is VII.

[0534] C109. The immunogenic composition of C108, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0535] C110. The immunogenic composition of C108 or C109, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0536] C111. The immunogenic composition of C68, wherein the at least one additional serotype is VIII.

[0537] C112. The immunogenic composition of C111, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0538] C113. The immunogenic composition of C112, wherein the at least one additional serotype is IX.

[0539] C114. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharides from GBS serotypes Ia, Ib, II, III and IV.

[0540] C115. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharides from GBS serotypes Ia, Ib, II, III and V.

[0541] C116. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharides from GBS serotypes Ia, Ib, II, III, IV and V.

[0542] C117. An immunogenic composition comprising a polysaccharide-protein conjugate comprising at least four GBS capsular polysaccharide serotypes selected from the group consisting of Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

[0543] C118. The immunogenic composition of C117, comprising at least five GBS capsular polysaccharide serotypes.

[0544] C119. The immunogenic composition of C117 or C118, comprising at least six GBS capsular polysaccharide serotypes.

[0545] C120. The immunogenic composition of any one of C117-C119, comprising at least seven GBS capsular polysaccharide serotypes.

[0546] C121. The immunogenic composition of any one of C117-C120, comprising at least eight GBS capsular polysaccharide serotypes.

[0547] C122. The immunogenic composition of any one of C117-C121, comprising at least nine GBS capsular polysaccharide serotypes.

[0548] C123. The immunogenic composition of any one of C117 to C122, comprising GBS capsular polysaccharide serotype V.

[0549] C124. The immunogenic composition of any one of C117 to C123, which does not have immune interference.

[0550] C125. The immunogenic composition of any one of C67-C124, further comprising a pharma- ceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic surfactant, inhibitor of free radical oxidation, carrier, or a mixture thereof.

[0551] C126. The immunogenic composition of any one of C67 to C125, further comprising a buffering agent.

[0552] C127. The immunogenic composition of C126, wherein the buffer is selected from the group consisting of HEPES, PIPES, MES, Tris(trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate.

[0553] C128. The immunogenic composition of C127, wherein the buffering agent is histidine.

[0554] C129. The immunogenic composition of any one of C67 to C128, further comprising a surfactant.

[0555] C130. World object.

[0556] C131. The immunogenic composition of C130, wherein the surfactant is polysorbate-80.

[0557] C132. The immunogenic composition of any one of C67 to C131, further comprising an excipient.

[0558] C133. The immunogenic composition of C132, wherein the excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol.

[0559] C134. The immunogenic composition of C133, wherein the excipient is sodium chloride.

[0560] C135. The immunogenic composition of any one of C67 to C134, further comprising an adjuvant.

[0561] C136. The immunogenic composition of C135, wherein the adjuvant is an aluminum-based adjuvant or QS-21.

[0562] C137. The immunogenic composition of C136, wherein the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyl phosphate and aluminum hydroxide.

[0563] C138. The immunogenic composition of C137, wherein the adjuvant is aluminum phosphate.

[0564] C139. The adjuvant is aluminum hydroxyl phosphate. 8. An immunogenic composition.

[0565] C140. The immunogenic composition of any one of C67-C139, comprising a buffer, a surfactant, an excipient, and optionally an adjuvant, and buffered to a pH of about 6.0 to about 7.0.

[0566] C141. The immunogenic composition of any one of C67-C140, comprising histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, buffered to a pH of about 6.0 to about 7.0.

[0567] C142. The immunogenic composition of any one of C67 to C141, comprising about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate.

[0568] C143. The immunogenic composition of any one of C67 to C142, comprising a dose of about 5 mcg / ml to about 50 mcg / ml.

[0569] C144. The immunogenic composition of any one of C67 to C143, optionally in the presence of at least one excipient, which is lyophilized.

[0570] C145. The immunogenic composition of C144, wherein at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol.

[0571] C146. The immunogenic composition of C145, wherein at least one excipient is sucrose.

[0572] C147. The immunogenic composition of any one of C144-C146, comprising about 1% (w / v) to about 10% (w / v) of at least one excipient.

[0573] C148. The immunogenic composition of any one of C144 to C147, comprising an additional excipient.

[0574] C149. The immunogenic composition of C148, wherein the additional excipient is mannitol or glycine.

[0575] C150. The immunogenic composition of C148 or C149, comprising about 1% (w / v) to about 10% (w / v) of an additional excipient.

[0576] C151. The immunogenic composition of any one of C143 to C150, which is reconstituted with water, water for injection (WFI), an adjuvant suspension or saline.

[0577] C152. The immunogenic composition of any one of C67 to C151 for use as a medicament.

[0578] C153. For use in a method for inducing an immune response against GBS in a subject The immunogenic composition according to any one of C67 to C152, for use in an immunological disease.

[0579] C154. The immunogenic composition of C153, wherein the subject is a woman planning pregnancy or who is pregnant.

[0580] C155. The immunogenic composition of C154, wherein the female is in the second half of pregnancy.

[0581] C156. The immunogenic composition of C155, wherein the pregnant female is at least 20 weeks pregnant.

[0582] C157. The immunogenic composition of C156, wherein the pregnant female is between 27 and 36 weeks pregnant.

[0583] C158. The immunogenic composition of C157, wherein the subject is an adult aged 50 years or older.

[0584] C159. The immunogenic composition of C158, wherein the subject is an adult aged 65 years or older.

[0585] C160. The immunogenic composition of C159, wherein the subject is an adult aged 85 years or older.

[0586] C161. The immunogenic composition of any one of C153 to C160, wherein the subject is immunocompromised.

[0587] C162. The immunogenic composition of C161, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

[0588] C163. The immunogenic composition of any one of C153 to C162, wherein the group B streptococcus is Streptococcus agalactiae.

[0589] C164. A method for inducing an immune response against group B streptococcus, comprising administering to a subject an effective amount of an immunogenic composition of any one of C67 to C150.

[0590] C165. A method of preventing or reducing a disease or condition associated with Group B Streptococcus in a subject, comprising administering to the subject an effective amount of an immunogenic composition of any one of C67 to C151.

[0591] C166. The method of C164 or C165, wherein the subject is a woman planning a pregnancy or a pregnant woman.

[0592] C167. The method of C166, wherein the woman is in the second half of pregnancy.

[0593] C168. A method of C166 or C167, wherein the pregnant woman is at least 20 weeks pregnant.

[0594] C169. The method of any one of C166 to C168, wherein the pregnant female is between 27 and 36 weeks pregnant.

[0595] C170. The method of C164 or C165, wherein the subject is an adult aged 50 years or older.

[0596] C171. The method of C170, wherein the subject is an adult aged 65 years or older.

[0597] C172. The method of C170 or C171, wherein the subject is an adult aged 85 years or older.

[0598] C173. The method of any one of C164 to C172, wherein the subject is immunocompromised.

[0599] C174. The method of C173, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

[0600] C175. The method of any one of C164 to C174, wherein the group B streptococcus is Streptococcus agalactiae.

[0601] C176. An antibody that binds to the capsular polysaccharide in the immunogenic conjugate of any one of C1-C49 or C66.

[0602] C177. A composition comprising the antibody of C176.

[0603] C178. A method for producing an antibody, comprising administering to a subject an immunogenic composition according to any one of C67 to C151.

[0604] C179. An antibody produced by the method of C178.

[0605] C180. A method for conferring passive immunity to a subject, comprising: (a) producing an antibody preparation using the immunogenic composition of any one of C67 to C151; (b) administering the antibody preparation to a subject to confer passive immunity; The method includes:

[0606] C181. A method for making an immunogenic polysaccharide-protein conjugate of any one of C1-C49 or C66, comprising: (a) reacting GBS capsular polysaccharide with an oxidizing agent to provide an activated polysaccharide; (b) reacting the activated polysaccharide with a carrier protein to provide a polysaccharide-protein conjugate; The method includes:

[0607] C182. The method of C181, wherein step (b) is carried out in a polar aprotic solvent.

[0608] C183. The method of C182, wherein the solvent is selected from the group consisting of dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF) and hexamethylphosphoramide (HMPA).

[0609] C184. The method of C183, wherein the solvent is dimethylsulfoxide (DMSO).

[0610] C185. The method of any one of C181-C184, wherein the polysaccharide is reacted with 0.01 to 10.0 molar equivalents of an oxidizing agent.

[0611] C186. The method of any one of C181-C185, wherein the oxidizing agent is periodate.

[0612] C187. The method of C186, wherein the periodate is sodium periodate.

[0613] C188. The method of any one of C181-C187, wherein the oxidation reaction of step (a) is for between 1 hour and 50 hours.

[0614] C189. The method of any one of C181-C188, wherein the temperature of the oxidation reaction is maintained between about 2° C. and about 25° C.

[0615] C190. The method of any one of C181-C189, wherein the oxidation reaction is carried out in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) and Bis-Tris.

[0616] C191. The method of C190, wherein the buffer has a concentration of between about 1 mM and about 500 mM.

[0617] C192. The method of any one of C181-C191, wherein the oxidation reaction is carried out at a pH between about 4.0 and about 8.0.

[0618] C193. The method of C181, wherein the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO).

[0619] C194. The method of C193, wherein N-chlorosuccinimide (NCS) is the co-oxidant.

[0620] C195. The method of any one of C181-C194, wherein step (a) further comprises quenching the oxidation reactant by addition of a quenching agent.

[0621] C196. The method of any one of C182-C195, wherein the concentration of the polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL.

[0622] C197. The method of any one of C181 to C196, wherein the degree of oxidation of the activated polysaccharide is between 5 and 25.

[0623] C198. The method of any one of C181-C197, further comprising the step of lyophilizing the activated polysaccharide.

[0624] C199. The method of C188, wherein the activated polysaccharide is lyophilized in the presence of a sugar selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit.

[0625] C200. Step (b) (1) combining an activated polysaccharide with a carrier protein; (2) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; The method of any one of C181 to C199, comprising:

[0626] C201. The method of C200, wherein the concentration of the activated polysaccharide in step (2) is between about 0.1 mg / mL and about 10.0 mg / mL.

[0627] C202. The initial ratio of activated polysaccharide to carrier protein (wt / wt) is 5:1 to 0. 1:1 between C200 or C201 methods.

[0628] C203. The process of any one of C200-C202, wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc in the presence of a Bronsted or Lewis acid, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMeiPrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB).

[0629] C204. The method of C203, wherein the reducing agent is sodium cyanoborohydride.

[0630] C205. The method of any one of C200-C204, wherein the amount of reducing agent is between about 0.1 and about 10.0 molar equivalents.

[0631] C206. The method of any one of C200 to C205, wherein the duration of the reduction reaction in step (2) is between 1 hour and 60 hours.

[0632] C207. The method of any one of C200-C206, wherein the temperature of the reduction reaction is maintained between 10° C. and 40° C.

[0633] C208. The method of any one of C181-C207, further comprising the step of capping any unreacted aldehyde by the addition of a borohydride (step (c)).

[0634] C209. The process of C208, wherein the amount of borohydride is between about 0.1 and about 10.0 molar equivalents.

[0635] C210. ​​The method of C208, wherein the borohydride is selected from the group consisting of sodium borohydride (NaBH4), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride and magnesium borohydride.

[0636] C211. The method of C208, wherein the borohydride is sodium borohydride (NaBH4).

[0637] C212. The method of any one of C207-C211, wherein the duration of the capping step is between 0.1 hours and 10 hours.

[0638] C213. The method of any one of C207-C212, wherein the temperature of the capping step is maintained between about 15° C. and about 45° C.

[0639] C214. The method of any one of C181-C213, further comprising purifying the polysaccharide-protein conjugate.

[0640] C215. The method of any one of C181-C214, wherein the polysaccharide-protein conjugate comprises less than about 40% free polysaccharide compared to the total amount of polysaccharide.

[0641] C216. The method of any one of C181-C215, wherein the ratio of polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.0.

[0642] C217. The degree of conjugation of the conjugate is between 2 and 15; The method according to any one of C181 to C216.

[0643] C218. A method for producing a polysaccharide-protein conjugate, comprising: (a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction of step (a) by the addition of a quenching agent to provide activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; (e) capping any unreacted aldehyde by addition of sodium borohydride (NaBH4); Including, wherein steps (c) and (d) are carried out in DMSO.

Claims

1. 1. An immunogenic polysaccharide-protein conjugate comprising a Group B Streptococcus (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%.

2. 2. The immunogenic conjugate of claim 1, wherein the capsular polysaccharide is selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

3. 3. The immunogenic conjugate of claim 1 or 2, wherein the capsular polysaccharide has at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 or 0.95 mM sialic acid per mM polysaccharide.

4. 4. The immunogenic conjugate of claim 1, wherein the capsular polysaccharide has a molecular weight of between about 5 kDa and about 1,000 kDa, between about 25 kDa and about 750 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 200 kDa, or between about 100 kDa and about 400 kDa.

5. 5. The immunogenic conjugate of claim 1, wherein the molecular weight of the conjugate is between about 300 kDa and about 20,000 kDa, between about 1,000 kDa and about 15,000 kDa, or between about 1,000 kDa and about 10,000 kDa.

6. 6. The immunogenic conjugate of claim 1, wherein the capsular polysaccharide is between about 0% and about 40% O-acetylated.

7. 7. The immunogenic conjugate of any one of claims 1 to 6, wherein the capsular polysaccharide is less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% O-acetylated.

8. 8. The immunogenic conjugate of claim 1, wherein the capsular polysaccharide has at least about 0.1, 0.2, 0.3, 0.35 or about 0.4 mM O-acetate per mM repeating saccharide unit.

9. 8. The immunogenic conjugate of claim 1, wherein the capsular polysaccharide has less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM repeating saccharide unit.

10. The carrier protein is CRM １９７ or tetanus toxoid.

11. The carrier protein is CRM １９７ The immunogenic conjugate of claim 10,

12. 1. A method for isolating capsular polysaccharide, comprising reacting an organic reagent with a cell broth containing a capsular polysaccharide-producing organism.

13. The method of claim 12, which does not lyse the bacteria.

14. 14. The method of claim 12 or 13, wherein the bacteria is killed by heat.

15. 15. The method of any one of claims 12 to 14, further comprising the step of centrifuging to provide a cell paste.

16. 16. The method of any one of claims 12 to 15, further comprising a filtering step.

17. 17. The method of claim 16, wherein the filtering step is hemodiafiltration.

18. Capsular polysaccharide-producing bacteria include Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and the like.

18. The method of any one of claims 12 to 17, wherein the bacterium is selected from the group consisting of Streptococcus faecium and Enterococcus faecalis.

19. 19. The method of claim 18, wherein the bacterium is Streptococcus agalactiae.

20. 20. The method of any one of claims 12 to 19, wherein the organic reagent is a derivatized hydroxylamine compound.

21. 21. The method of any one of claims 12 to 20, wherein the hydroxylamine is any hydroxylamine listed in Table 2 of Example 2.

22. 22. The method of any one of claims 12 to 21, wherein the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine, diethylhydroxylamine, hydroxylamine, ethylenediamine, triethylenetetramine, 1,1,4,7,10,10 hexamethyltriethylenetetramine and 2,6,10,trimethyl 2,6,10 triazaundecane.

23. 23. The method of any one of claims 12 to 22, wherein the concentration of hydroxylamine is from about 5 mM to about 200 mM.

24. 24. The method of any one of claims 12 to 23, wherein the pH of the reaction is from about 5.5 to about 9.

5.

25. 25. The method of any one of claims 12 to 24, wherein the reaction occurs at a temperature of from about 20°C to about 85°C.

26. 26. The method of any one of claims 12 to 25, wherein the reaction reaction time is from about 10 hours to about 90 hours.

27. A method for producing an immunogenic polysaccharide-protein conjugate according to any one of claims 1 to 11, wherein the capsular polysaccharide is isolated according to a method according to any one of claims 12 to 26.

28. 27. An immunogenic polysaccharide-protein conjugate comprising a capsular polysaccharide prepared by the method of any one of claims 12 to 26.

29. An immunogenic composition comprising an immunogenic polysaccharide-protein conjugate according to any one of claims 1 to 11 or claim 28.

30. 1. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharide from Group B Streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

31. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharides from GBS serotypes Ia, Ib, II, III and IV.

32. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharides from GBS serotypes Ia, Ib, II, III and V.

33. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharides from GBS serotypes Ia, Ib, II, III, IV and V.

34. 1. An immunogenic composition comprising a polysaccharide-protein conjugate comprising at least four GBS capsular polysaccharide serotypes selected from the group consisting of Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

35. 35. The immunogenic composition of claim 34, comprising GBS capsular polysaccharide serotype V.

36. 36. The immunogenic composition of claim 34 or 35, which is free of immune interference.

37. 37. The immunogenic composition of any one of claims 29 to 36, further comprising a pharma- ceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic surfactant, inhibitor of free radical oxidation, carrier, or a mixture thereof.

38. 38. The immunogenic composition of any one of claims 29 to 37, further comprising a buffering agent.

39. 39. The immunogenic composition of claim 38, wherein the buffer is selected from the group consisting of HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate.

40. 40. The immunogenic composition of claim 39, wherein the buffer is histidine.

41. 41. The immunogenic composition of any one of claims 29 to 40, further comprising a surfactant.

42. The immunogenic composition of claim 41, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20 and polyoxyethylene alkyl ethers.

43. The immunogenic composition of claim 42, wherein the surfactant is polysorbate-80.

44. 44. The immunogenic composition of any one of claims 29 to 43, further comprising an excipient.

45. 45. The immunogenic composition of claim 44, wherein the excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol.

46. 46. ​​The immunogenic composition of claim 45, wherein the excipient is sodium chloride.

47. 47. The immunogenic composition of any one of claims 29 to 46, further comprising an adjuvant.

48. The immunogenic composition of claim 47, wherein the adjuvant is an aluminum-based adjuvant or QS-21.

49. 49. The immunogenic composition of claim 48, wherein the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyl phosphate and aluminum hydroxide.

50. 50. The immunogenic composition of claim 49, wherein the adjuvant is aluminum phosphate.

51. 50. The immunogenic composition of claim 49, wherein the adjuvant is aluminum hydroxyl phosphate.

52. 52. The immunogenic composition of any one of claims 29 to 51, comprising a buffer, a surfactant, an excipient, and optionally an adjuvant, and buffered to a pH of about 6.0 to about 7.

0.

53. 53. The immunogenic composition of any one of claims 29 to 52, comprising histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, buffered to a pH of about 6.0 to about 7.

0.

54. 54. The immunogenic composition of any one of claims 29 to 53, comprising about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate.

55. 55. The immunogenic composition of any one of claims 29 to 54 comprising a dose of about 5mcg / ml to about 50mcg / ml.

56. 56. The immunogenic composition of any one of claims 29 to 55, which is lyophilized, optionally in the presence of at least one excipient.

57. 57. The immunogenic composition of claim 56, wherein at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol.

58. 58. The immunogenic composition of claim 57, wherein at least one excipient is sucrose.

59. 59. The immunogenic composition of any one of claims 56 to 58, comprising from about 1% (w / v) to about 10% (w / v) of at least one excipient.

60. 60. The immunogenic composition of any one of claims 56 to 59, comprising an additional excipient.

61. 61. The immunogenic composition of claim 60, wherein the additional excipient is mannitol or glycine.

62. 62. The immunogenic composition of claim 60 or 61, comprising about 1% (w / v) to about 10% (w / v) of an additional excipient.

63. 63. The immunogenic composition of any one of claims 29 to 62, which is reconstituted with water, water for injection (WFI), an adjuvant suspension or saline.

64. 64. An immunogenic composition according to any one of claims 29 to 63 for use as a medicament.

65. 64. An immunogenic composition according to any one of claims 29 to 63 for use in a method of inducing an immune response against GBS in a subject.

66. The immunogenic composition of claim 65, wherein the subject is a woman who is planning a pregnancy or is pregnant.

67. 67. The immunogenic composition of claim 66, wherein the woman is in the second half of pregnancy, at least 20 weeks pregnant, or between 27 and 36 weeks pregnant.

68. 66. The immunogenic composition of claim 65, wherein the subject is an adult aged 50 years or older, 65 years or older, or 85 years or older.

69. 69. The immunogenic composition of any one of claims 65 to 68, wherein the subject is immunocompromised.

70. 70. The immunogenic composition of claim 69, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

71. 71. The immunogenic composition of any one of claims 65 to 70, wherein the group B streptococcus is Streptococcus agalactiae.

72. 64. A method for inducing an immune response against group B Streptococcus, comprising administering to a subject an effective amount of an immunogenic composition described in any one of claims 29 to 63.

73. A method for preventing or reducing a disease or condition associated with Group B Streptococcus in a subject, comprising administering to the subject an effective amount of an immunogenic composition described in any one of claims 29 to 63.

74. 74. The method of claim 72 or 73, wherein the subject is a woman who is planning a pregnancy or is pregnant.

75. 75. The method of claim 74, wherein the woman is in the second half of pregnancy, at least 20 weeks pregnant, or between 27 and 36 weeks pregnant.

76. 74. The method of claim 72 or 73, wherein the subject is an adult aged 50 years or older, 65 years or older, or 85 years or older.

77. 77. The method of any one of claims 72 to 76, wherein the subject is immunocompromised.

78. 78. The method of claim 77, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

79. 79. The method of any one of claims 72 to 78, wherein the group B streptococcus is Streptococcus agalactiae.

80. 30. An immunogenic conjugate according to any one of claims 1 to 11 or 28, wherein the antibody binds to the capsular polysaccharide.

81. A composition comprising the antibody of claim 80.

82. 64. A method for producing antibodies, comprising administering to a subject an immunogenic composition according to any one of claims 29 to 63.

83. 83. An antibody produced by the method of claim 82.

84. 1. A method of conferring passive immunity to a subject, comprising: (a) producing an antibody preparation using the immunogenic composition of any one of claims 29 to 63; (b) administering the antibody preparation to a subject to confer passive immunity; The method includes:

85. A method for producing an immunogenic polysaccharide-protein conjugate according to any one of claims 1 to 11 or claim 28, comprising the steps of: (a) reacting GBS capsular polysaccharide with an oxidizing agent to provide an activated polysaccharide; (b) reacting the activated polysaccharide with a carrier protein to provide a polysaccharide-protein conjugate; The method includes:

86. 86. The method of claim 85, wherein step (b) is carried out in a polar aprotic solvent.

87. 87. The method of claim 86, wherein the solvent is selected from the group consisting of dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF) and hexamethylphosphoramide (HMPA).

88. 88. The method of claim 87, wherein the solvent is dimethylsulfoxide (DMSO).

89. 89. The method of any one of claims 85 to 88, wherein the polysaccharide is reacted with 0.01 to 10.0 molar equivalents of the oxidizing agent.

90. 90. The method of any one of claims 85 to 89, wherein the oxidizing agent is periodate.

91. 91. The method of claim 90, wherein the periodate is sodium periodate.

92. 92. The method of any one of claims 85 to 91, wherein the oxidation reaction of step (a) is for between 1 hour and 50 hours.

93. 93. The method of any one of claims 85 to 92, wherein the temperature of the oxidation reaction is maintained between about 2°C and about 25°C.

94. 94. The method of any one of claims 85 to 93, wherein the oxidation reaction is carried out in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) and Bis-Tris.

95. 95. The method of claim 94, wherein the buffering agent has a concentration of between about 1 mM and about 500 mM.

96. 96. The method of any one of claims 85 to 95, wherein the oxidation reaction is carried out at a pH between about 4.0 and about 8.

0.

97. 86. The method of claim 85, wherein the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO).

98. 98. The method of claim 97, wherein N-chlorosuccinimide (NCS) is the co-oxidant.

99. 99. The method of any one of claims 85 to 98, wherein step (a) further comprises quenching the oxidation reactant by addition of a quenching agent.

100. 100. The method of any one of claims 85 to 99, wherein the concentration of the polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL.

101. 101. The method of any one of claims 85 to 100, wherein the degree of oxidation of the activated polysaccharide is between 5 and 25.

102. 102. The method of any one of claims 85 to 101, further comprising the step of lyophilizing the activated polysaccharide.

103. 103. The method of claim 102, wherein the activated polysaccharide is lyophilized in the presence of a sugar selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit.

104. Step (b) (1) combining an activated polysaccharide with a carrier protein; (2) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; 104. The method of any one of claims 85 to 103, comprising:

105. 105. The method of claim 104, wherein the concentration of the activated polysaccharide in step (2) is between about 0.1 mg / mL and about 10.0 mg / mL.

106. 106. The method of claim 104 or 105, wherein the initial ratio (w / w) of activated polysaccharide to carrier protein is between 5:1 and 0.1:

1.

107. The reducing agent is a Bronsted or Lewis acid, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe ｉ PrN-BH ３ , benzylamine-BH ３ or 5-ethyl-2-methylpyridine borane (PEMB), wherein the reactant is selected from the group consisting of sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride and zinc.

108. 108. The method of claim 107, wherein the reducing agent is sodium cyanoborohydride.

109. 109. The method of any one of claims 104 to 108, wherein the amount of reducing agent is between about 0.1 and about 10.0 molar equivalents.

110. 110. The method of any one of claims 104 to 109, wherein the duration of the reduction reaction in step (2) is between 1 hour and 60 hours.

111. 111. The method of any one of claims 104 to 110, wherein the temperature of the reduction reaction is maintained between 10°C and 40°C.

112. 112. The method of any one of claims 104 to 111, further comprising the step of capping any unreacted aldehyde by the addition of a borohydride (step (c)).

113. 113. The method of claim 112, wherein the amount of borohydride is between about 0.1 and about 10.0 molar equivalents.

114. The borohydride is sodium borohydride (NaBH ４ ), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride and magnesium borohydride.

115. The borohydride is sodium borohydride (NaBH ４ ). The method of claim 114 .

116. 115. The method of any one of claims 112 to 114, wherein the duration of the capping step is between 0.1 hours and 10 hours.

117. 117. The method of any one of claims 112 to 116, wherein the temperature of the capping step is maintained between about 15°C and about 45°C.

118. 118. The method of any one of claims 85 to 117, further comprising the step of purifying the polysaccharide-protein conjugate.

119. 119. The method of any one of claims 85 to 118, wherein the polysaccharide-protein conjugate comprises less than about 40% free polysaccharide compared to the total amount of polysaccharide.

120. 120. The method of any one of claims 85 to 119, wherein the ratio of polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.

0.

121. 121. The method of any one of claims 85 to 120, wherein the degree of conjugation of the conjugate is between 2 and 15.

122. 1. A method for making a polysaccharide-protein conjugate, comprising: (a) reacting isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction of step (a) by the addition of a quenching agent to provide activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; (e) Sodium borohydride (NaBH ４ capping any unreacted aldehyde by the addition of Including, The method, wherein steps (c) and (d) are carried out in DMSO.

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