Group B streptococcal polysaccharide-protein conjugate, method for producing the conjugate, immunogenic composition containing the conjugate, and uses thereof

Abstract

The present invention relates to an immunogenic polysaccharide-protein conjugate comprising a capsular polysaccharide (CP) from Streptococcus agalactiae (commonly known 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 wherein the sialic acid level of the CP is higher than about 60%. The present invention also relates to methods of making the conjugate and immunogenic compositions comprising the conjugate. The present invention also relates to an immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises CP from GBS serotype IV and at least one additional serotype. The present invention further relates to methods of using the compositions disclosed herein to induce an immune response against GBS in a subject and / or to reduce or prevent invasive GBS disease in a subject. The antibodies generated can be used for the treatment or prevention of GBS infection by passive immunotherapy.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680025709.4, with an application date of April 29, 2016 and an invention title of "Group B Streptococcus Polysaccharide-Protein Conjugate, Method for Producing the Conjugate, Immunogenic Composition Containing the Conjugate and Use Thereof". Technical Field

[0002] The present invention relates to immunogenic polysaccharide-protein conjugates comprising capsular polysaccharide (CP) from Streptococcus agalactiae (commonly known 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 wherein the amount of sialic acid in the CP is higher than about 60%. The present invention also relates to a method for producing the conjugate and an immunogenic composition comprising the conjugate. The present invention also relates to an immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises CP from GBS serotype IV and at least one additional serotype. The present invention further relates to a method of using the compositions disclosed herein to induce an immune response against GBS in a subject and / or reduce or prevent invasive GBS disease in a subject. The antibodies produced can be used for the treatment or prevention of GBS infection by passive immunotherapy. Background of the Invention

[0003] Streptococcus agalactiae is a Gram-positive polysaccharide-encapsulated organism, also known as group B streptococcus (GBS). They are common commensals of the human gastrointestinal and genital tracts and are also a cause of serious disease in infants and the elderly (Baker, C.J., Vaccine, 31(Suppl. 4): D3-D6 (2013)). The main risk factor for GBS infection in infants is maternal colonization (Dillon, H.C., et al., J. Pediatr., 110(1): 31-36 (1987)). Up to a quarter of women carry GBS in their rectovagina, and this GBS can infect the amniotic fluid or the infant before or during delivery, causing sepsis, pneumonia, and meningitis (Baker 2013; Heath, P.T., et al., BMJ Clin. Evid. (Online), pii: 0323 (2014)). Twenty-five percent of infants who survive GBS meningitis have neurological damage, and 19% of them experience cognitive delay, cerebral palsy, blindness, and hearing loss (Libster, R., et al., Pediatrics, 130(1): e8-152012 (2012)). GBS can also cause miscarriage and preterm birth and is associated with stillbirth (McDonald, H.M., et al., Infectious Diseases in Obstetrics and Gynecology, 8(5-6): 220-227 (2000); Randis, T.M., 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)). The risk of infection in very low birth weight infants is much higher, with an infection rate of up to 3% and a mortality rate of up to 30% (even with immediate antibiotic treatment) (Heath 2014).

[0004] The introduction of GBS screening and intrapartum antibiotic prophylaxis (IAP) in the United States in the late 1990s has proven to reduce the rate of neonatal diseases (early-onset diseases [EOD]) occurring within the first week of birth, but has no measurable effect on the rate of late-onset diseases (LOD) occurring within the first 3 months after birth. The current rates of EOD and LOD cases in the United States are 0.25 and 0.27 per 1000 births, respectively (Centers for Disease Control and Prevention (CDC), Active Bacterial Core Surveillance Report (2013), available at http: / / www.cdc.gov / abcs / reports-findings / survreports / gbs13.pdf). After the introduction of pneumococcal conjugate vaccines for the prevention of invasive pneumococcal diseases (including bacteremia and meningitis), GBS has become the most common single cause of neonatal sepsis (EOD) and meningitis (<2 mo) in the United States, despite the availability of IAP for the prevention of GBS disease (Verani, J.R., et al., MMWR, 59(RR10):1-32(2010); Thigpen, M.C., et al., New England Journal of Medicine, 364(21):2016-2025(2011)). Unlike in the United States, the introduction of guidelines for the prevention and control of invasive GBS disease and IAP has not reduced the incidence of EOD in the Netherlands or the United Kingdom (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 comprehensive screening and the limitation of IAP to mothers in the highest risk groups (e.g., fever, prolonged rupture of membranes). The proportion of EOD in countries where IAP is not used is significantly higher, with an average reported incidence of 0.75 per 1000 live births (95% CI 0.58-0.89) (Edmond, K.M, et al., Lancet, 379(9815):547-556(2012)).

[0005] Another group at risk for GBS disease is the elderly. Risk factors include chronic medical problems such as diabetes, cancer, heart failure, and neurological and urinary tract conditions. According to CDC ABC surveillance data, the annual incidence of invasive GBS in the United States in 2013 was 0.28 / 1,000 adults or 12,400 cases per year in adults ≥65 years of age. This rate is similar to the incidence of invasive pneumococcal disease in the elderly (0.30 / 1,000 for >65). These rates are expected to continue to increase in the United States and Europe (CDC 2013; Lamagni 2013).

[0006] One approach to preventing GBS disease in infants and the elderly is the use of polysaccharide-based vaccines. In the United States, administration of maternal GBS prophylactic vaccines has the potential to prevent GBS disease in infants, regardless of whether IAP is used. Although polysaccharides themselves can be immunogenic, conjugation of polysaccharides to protein carriers has been used to improve immunogenicity, particularly for infants and the elderly. Polysaccharide-protein conjugate vaccines are made using polysaccharides (usually from the surface layer of bacteria) linked to protein carriers. Chemical bonding of the polysaccharide to the protein carrier can induce an immune response against bacteria that display the polysaccharide contained in the vaccine on their surface, thereby preventing disease. Thus, vaccination with polysaccharides from pathogenic bacteria is a possible strategy for enhancing host immunity.

[0007] The polysaccharides that coat bacteria vary widely, even within a single species. For example, there are ten different serotypes in GBS due to variation in the bacterial polysaccharide capsule. Thus, it is desirable for polysaccharide-based vaccines to consist of a group of polysaccharides to ensure broad coverage of different epidemic strains.

[0008] The carrier protein can be a related protein antigen from the target pathogen, enhancing the specific immune response against that pathogen, or it can be a generally immunogenic protein that also acts as an adjuvant or general immune response stimulant.

[0009] Individual monovalent polysaccharide-protein conjugates of GBS serotypes Ia, Ib, II, III, and V have been evaluated in Phase 1 and Phase 2 clinical trials in non-pregnant adults (Brigtsen, A.K., et al., Journal of Infectious Diseases, 185(9):1277-1284 (2002); Baker, C.J., et al., J. Infect. Dis., 188(1):66-73 (2003); Baker, C.J., et al., J. Infect. Dis., 189(6):1103-1112 (2004); Baker, C.J., et al., Vaccine, 25(1):55-63 (2007)). Bivalent II-TT and III-TT glycoconjugate vaccines, as well as a trivalent vaccine including Ia-CRM 197 , Ib-CRM 197 , and III-CRM 197 conjugates have also been studied (Baker JID 2003; Clinicaltrials.gov NCT01193920, NCT01412801, and NCT01446289). However, no GBS vaccine has been approved.

[0010] In addition, although the trivalent vaccine covers >90% of the invasive strains causing neonatal disease in South Africa (Madzivhandila, M., et al., PloS One, 6(3):e17861 (2011)), based on a recent surveillance of neonatal isolates from 901 global collections from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T., http: / / www.testsurveillance.com / ) collected from 2004 to 2013, these same serotypes represent only 62% and 66% of the invasive isolates in the North American and European regions, respectively.

[0011] Analysis of strains obtained from T.E.S.T. samples revealed that 95% of the strains collected belonged to one of five well-documented major serotypes (Ia, Ib, II, III, and V), while an additional 3% were serotype IV. A series of publications have also confirmed the emergence of serotype IV in the Americas and Europe over the past decade (Diedrick, M.J., 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., EuroSurveillance: Bulletin European sur les Maladies Transmissibles (European Communicable Disease Bulletin), 19(23) (2014); Palmiero, J.K., et al., Journal of Clinical Microbiology, 48(12):4397-4403 (2010)). Studies investigating adult rectal / vaginal carriage (a risk factor for transmission of GBS to infants) have also found that 97% of isolates belong to one of these six serotypes, with serotype IV representing ~4% of the frequency. This study was designed to monitor carriage of beta-hemolytic streptococci (which includes GBS), Clostridium difficile, and Staphylococcus aureus in healthy adults in the United States (see Matson, M.A., et al., ICAAC, Abstract I-306 (Washington, DC, Sep. 5-9, 2014)).

[0012] Similarly, analysis of T.E.S.T. samples showed that 98% of blood isolates from older US adults ≥65 years of age belonged to the same six major serogroups. The most obvious difference between isolates from the elderly and other groups was serogroup distribution. With respect to isolates from elderly patients, serotype V strains constituted the largest group (34%, compared to 18% of neonatal carriage strains, or 18% of adult carriage strains).

[0013] Other studies have found regional variation in serotype prevalence. For example, serotype VI and VIII isolates have been identified as the major colonizers of healthy pregnant women in Japan (Lachenauer, C.S., et al., JID 179(4):1030-1033 (1999)).

[0014] Accordingly, there is a need for polysaccharide-protein conjugate vaccines or monoclonal antibodies to confer passive immunity as a means of preventing or treating GBS disease in a large segment of the worldwide population, including those caused by the emerging serotype IV. SUMMARY OF THE INVENTION

[0015] The present invention relates to novel immunogenic GBS polysaccharide-protein conjugates, methods of making the conjugates, and immunogenic compositions comprising the conjugates, and includes the inventions disclosed in U.S. Provisional Application No. 62 / 156,500, filed May 4, 2015, U.S. Provisional Application No. 62 / 237,813, filed May 4, 2015, and U.S. Provisional Application No. 62 / 237,820, filed October 6, 2015, the entire contents of which are incorporated herein by reference. The following describes certain aspects and embodiments of the invention.

[0016] 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 sialic acid level of the capsular polysaccharide is greater than about 60%, greater than about 95%, or about 100%. In another embodiment, the capsular polysaccharide can be desialylated to up to about 40% (sialylation level 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.

[0017] In a further aspect, the immunogenic polysaccharide-protein conjugate comprises a capsular polysaccharide having about 1.0 mM of sialic acid per mM of polysaccharide, such as having at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 mM of sialic acid per mM of polysaccharide.

[0018] In another aspect of the invention, the immunogenic conjugate comprises a capsular polysaccharide having a molecular weight of about 5 kDa to about 1,000 kDa, about 25 kDa to about 750 kDa, about 25 kDa to about 400 kDa, about 25 kDa to about 200 kDa, or about 100 kDa to about 400 kDa.

[0019] In additional embodiments, the immunogenic conjugate of the invention has a molecular weight of from about 300 kDa to about 20,000 kDa, such as from about 1,000 kDa to about 15,000 kDa, or from about 1,000 kDa to about 10,000 kDa.

[0020] In one embodiment, the immunogenic conjugate comprises a capsular polysaccharide that is from about 0% to about 40% O-acetylated, such as less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% O-acetylated.

[0021] In one embodiment, the immunogenic conjugate comprises a capsular polysaccharide that has at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM O-acetate per mM sugar repeat unit. In another embodiment, the immunogenic conjugate comprises a capsular polysaccharide that has less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM sugar repeat unit.

[0022] In one embodiment, the immunogenic conjugate comprises CRM 197 or tetanus toxoid as a carrier protein. In certain embodiments, the carrier protein is CRM 197 .

[0023] Another aspect of the present invention relates to a method for separating capsular polysaccharides, which comprises reacting an organic reagent with a cell broth containing bacteria producing capsular polysaccharides. In one embodiment, the method further comprises a centrifugation step. In another embodiment, the method further comprises a filtration step. In a particular embodiment, the bacteria producing capsular polysaccharides are 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 of amines listed in Table 2. In another embodiment, the hydroxylamine is selected from the group consisting of: dibenzyl hydroxylamine; diethylhydroxylamine; hydroxylamine; ethylenediamine; triethylenetetramine; 1,1,4,7,10,10 hexamethyltriethylene tetramine; and 2,6,10, Trimethyl 2,6,10 triazaundecane. In another embodiment, the concentration of the hydroxylamine is from about 5 mM to about 200 mM. In another embodiment, the pH of the reaction is from about 5.5 to about 9.5. In another embodiment, the reaction is carried out at a temperature of about 20 °C to about 85 °C. In another embodiment, the reaction time is from about 10 hours to about 90 hours.

[0024] In one aspect, the present invention relates to an immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae (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 a particular embodiment, the conjugate comprises capsular polysaccharides from serotypes Ia, Ib, II, III, and IV. In another embodiment, the composition comprises GBS serotype V. In a particular embodiment, the conjugate comprises capsular polysaccharides from serotypes Ia, Ib, II, III, and V. In another embodiment, the immunogenic composition comprises six polysaccharide-protein conjugates, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae 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.

[0025] In one embodiment, the immunogenic composition further comprises a pharmaceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, nonionic detergent, free radical oxidation inhibitor, carrier, or a mixture thereof. In a further embodiment, a buffer is included. The buffer can be HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine, or succinate. In a preferred embodiment, the buffer is histidine.

[0026] In another embodiment, the immunogenic composition further comprises a surfactant. The surfactant can be polyoxyethylene sorbitan fatty acid ester, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20, or polyoxyethylene alkyl ether. In a preferred embodiment, the surfactant is polysorbate-80.

[0027] In another 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, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim milk powder, glycerol, propylene glycol, water, and ethanol. In a preferred embodiment, the excipient is sodium chloride.

[0028] In 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, hydroxyaluminum phosphate, and aluminum hydroxide. In a more preferred embodiment, the adjuvant is aluminum phosphate.

[0029] In one aspect of the invention, the immunogenic composition comprises a buffer, a surfactant, an excipient, and an optional adjuvant, wherein the composition is buffered to a pH of from about 6.0 to about 7.0. In another aspect, the immunogenic composition comprises histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the composition is buffered to a pH of from about 6.0 to about 7.0. In a preferred embodiment, the immunogenic composition comprises from about 10 mM to about 25 mM of histidine, from about 0.01% to about 0.03% (v / w) of polysorbate-80, from about 10 mM to about 250 mM of sodium chloride (NaCl), and optionally from about 0.25 mg / ml to about 0.75 mg / ml of aluminum as aluminum phosphate. In another aspect of the invention, the immunogenic composition comprises a dose of from about 5 mcg / ml to about 50 mcg / ml.

[0030] In another aspect of the present invention, the immunogenic composition, optionally in the presence of at least one excipient, is lyophilized. 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, palatinitol, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim 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 from about 1% (w / v) to about 10% (w / v) of at least one excipient, preferably more than about 5.5% (w / v). In another embodiment, the lyophilized composition comprises additional excipients. In one such embodiment, the additional excipient is mannitol or glycine. In a preferred embodiment, the lyophilized composition comprises from about 1% (w / v) to about 10% (w / v) of the additional excipient. In another embodiment, the lyophilized composition is reconstituted with water, water for injection (WFI), 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.

[0031] Another aspect of the present invention relates to a method of inducing an immune response against GBS, which comprises administering to a subject an effective amount of an immunogenic composition as described herein. In one embodiment, the present invention relates to a method of preventing or alleviating a GBS-related disease or condition in a subject, which comprises administering to the subject an effective amount of an immunogenic composition as described herein. In a particular embodiment, the subject is a female planning to become pregnant or a pregnant female. In one such embodiment, the pregnant female is in the second half of pregnancy, such as at least at 20 weeks of gestation or at least at 27 weeks of gestation. In a preferred embodiment, the pregnant female is at 27 to 36 weeks of gestation. In another embodiment, the subject is an older adult, such as an adult 50 years of age or older, 65 years of age or older, and 85 years of age or older. In another 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.

[0032] Another aspect of the invention relates to antibodies that bind to the capsular polysaccharides in the immunogenic compositions of the invention. In some embodiments, the antibodies are generated upon administration of the immunogenic composition to a subject. Another aspect relates to compositions comprising the antibodies of the invention.

[0033] Another aspect of the invention relates to a method of conferring passive immunity to a subject, the method comprising the steps of: (a) generating an antibody preparation using the immunogenic composition described herein; and (b) administering the antibody preparation to the subject to confer passive immunity.

[0034] One aspect of the invention relates to a method of making the immunogenic polysaccharide-protein conjugate of the invention, the method comprising the steps of: (a) reacting a GBS capsular polysaccharide with an oxidizing agent to produce an activated polysaccharide; and (b) reacting the activated polysaccharide with a carrier protein to produce a polysaccharide-protein conjugate, wherein step (b) is carried out in a polar aprotic solvent. The solvent can be dimethyl sulfoxide (DMSO), sulfolane, dimethylformamide (DMF), and hexamethylphosphoramide (HMPA). In a preferred embodiment, the solvent is dimethyl sulfoxide (DMSO).

[0035] In one embodiment, the polysaccharide is reacted with 0.01 to 10.0 molar equivalents of the oxidizing agent. In a particular embodiment, the oxidizing agent is periodate. In such an embodiment, the periodate is sodium periodate.

[0036] In another embodiment, the oxidation reaction is from 1 hour to 50 hours. In another embodiment, the temperature of the oxidation reaction is maintained at about 2°C to about 25°C. In another embodiment, 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 such an embodiment, the concentration of the buffer is about 1 mM to about 500 mM. In a particular embodiment, the oxidation reaction is carried out at a pH of about 4.0 to about 8.0.

[0037] In another aspect of the invention, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). In such an embodiment, N-chlorosuccinimide (NCS) is a co-oxidizing agent.

[0038] In one embodiment, step (a) of the method of making the immunogenic polysaccharide-protein conjugate of the invention further comprises quenching the oxidation reaction by adding a quenching agent.

[0039] In another embodiment, the concentration of the polysaccharide is from about 0.1 mg / mL to about 10.0 mg / mL.

[0040] In a further embodiment, the degree of oxidation (DO) of the activated polysaccharide is 5 to 25.

[0041] In another aspect of the present 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 pinitol.

[0042] In another aspect of the present invention, step (b) of the method for producing an immunogenic polysaccharide-protein conjugate comprises: (1) mixing the activated polysaccharide with a carrier protein, and (2) reacting the mixed 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 from about 0.1 mg / mL to about 10.0 mg / mL. In a further embodiment, the initial ratio (weight / weight) of the activated polysaccharide to the carrier protein is 5:1 to 0.1:1. In another embodiment, the reducing agent is selected from the group consisting of sodium cyanoborohydride (NaBH 3 CN), sodium triacetoxyborohydride, sodium borohydride and zinc borohydride in the presence of a Bronsted acid or a Lewis acid, amine boranes such as pyridine borane, 2-methylpyridine borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i PrN-BH 3 、benzylamine-BH 3 or 5-ethyl-2-methylpyridine borane (PEMB). In a preferred embodiment, the reducing agent is (NaBH 3 CN). In another embodiment, the amount of the reducing agent is from about 0.1 to about 10.0 molar equivalents. In another embodiment, the duration of the reduction reaction in step (2) is 1 hour to 60 hours. In another embodiment, the temperature of the reduction reaction is maintained at 10°C to 40°C.

[0043] In a further aspect of the present invention, the method for producing an immunogenic polysaccharide-protein conjugate further comprises the step of capping unreacted aldehydes by adding borohydride (step (c)). In one embodiment, the amount of borohydride is from about 0.1 to about 10.0 molar equivalents. In another embodiment, the borohydride is selected from the group consisting of sodium borohydride (NaBH 4) Sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, and magnesium borohydride. In a preferred embodiment, the borohydride is sodium borohydride (NaBH 4 ). In another embodiment, the duration of the capping step is from 0.1 hour to 10 hours. In another embodiment, the temperature of the capping step is maintained at about 15°C to about 45°C.

[0044] In another aspect of the present invention, the method further comprises the step of purifying the polysaccharide-protein conjugate. In one embodiment, the polysaccharide-protein conjugate contains less than about 40% free polysaccharide compared to the total amount of polysaccharide. In another embodiment, the ratio (weight / weight) of polysaccharide to carrier protein in the conjugate is about 0.5 to about 3.0. In another embodiment, the degree of conjugation of the conjugate is 2 to 15.

[0045] In another aspect of the present invention, a method for manufacturing the immunogenic polysaccharide-protein conjugate of the present invention comprises the following steps: (a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction of step (a) by adding a quenching agent to produce an activated GBS capsular polysaccharide; (c) mixing the activated GBS capsular polysaccharide with a carrier protein, (d) reacting the mixed activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate, and (e) capping unreacted aldehydes by adding sodium borohydride (NaBH 4 ), wherein steps (c), (d), and (e) are carried out in DMSO. Brief Description of the Drawings

[0046] Figure 1 Compare the opsonophagocytic activity of sera and isolated IgG from mice immunized with monovalent vaccines of GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBSIV-CRM 197 and GBS V-CRM 197 .

[0047] Figure 2 GBS Ia-CRM 197 Stability after accelerated storage (4 weeks) at 50°C (as shown by % change in molecular weight, using SEC MALLS).

[0048] Figure 3 GBS Ib-CRM 197Stability after accelerated storage at 50°C (for 4 weeks) (as shown by % change in molecular weight, using SEC MALLS).

[0049] Figure 4 GBS II-CRM 197 Stability after accelerated storage at 50°C (for 4 weeks) (as shown by % change in molecular weight, using SEC MALLS).

[0050] Figure 5 GBS III-CRM 197 Stability after accelerated storage at 50°C (for 4 weeks) (as shown by % change in molecular weight, using SEC MALLS).

[0051] Figure 6 GBS IV-CRM 197 Stability after accelerated storage at 50°C (for 4 weeks) (as shown by % change in molecular weight, using SEC MALLS).

[0052] Figure 7 GBS V-CRM 197 Stability after accelerated storage at 50°C (for 4 weeks) (as shown by % change in molecular weight, using SEC MALLS).

[0053] Figure 8 GBS Ia-CRM 197 GBS Ib-CRM 197 GBS III-CRM 197 GBS IV-CRM 197 Stability after storage at 37°C (as shown by free sialic acid).

[0054] Figure 9 Stability of the hexavalent GBS vaccine using succinate as buffer (GBS Ia-CRM 197 GBS Ib-CRM 197 GBSII-CRM 197 GBS III-CRM 197 GBS IV-CRM 197 GBS V-CRM 197 ) in terms of pH.

[0055] Figure 10 Stability of the hexavalent GBS vaccine using histidine as buffer (GBS Ia-CRM 197 GBS Ib-CRM 197 GBSII-CRM 197 GBS III-CRM 197 GBS IV-CRM197 and GBS V-CRM 197 ) pH stability.

[0056] Figure 11 Hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) Effect of histidine buffer concentration in the hexavalent GBS vaccine on the binding of the GBS conjugate to aluminum (dose 10 mcg / ml).

[0057] Figure 12 Hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) Effect of histidine buffer concentration in the hexavalent GBS vaccine on the binding of the GBS conjugate to aluminum (dose 40 mcg / ml).

[0058] Figure 13 Hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) Effect of polysorbate-80 concentration in the hexavalent GBS vaccine on the percentage loss of total antigenicity under agitation pressure.

[0059] Figure 14 Hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) Effect of aluminum concentration in the hexavalent GBS vaccine on the binding of the GBS conjugate to aluminum.

[0060] Figure 15Effect of 5.5% (w / v) sucrose in freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on antigenicity recovery of each serotype.

[0061] Figure 16 Effect of 7.0% (w / v) sucrose in freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on antigenicity recovery of each serotype.

[0062] Figure 17 Effect of 8.5% (w / v) sucrose in freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on antigenicity recovery of each serotype.

[0063] Figure 18 Effect of 5.5% (w / v) sucrose in freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on antigenicity recovery of each serotype.

[0064] Figure 19 Effect of 5.5% (w / v) sucrose in freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197, GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on the antigenicity recovery of each serotype with 7.0% (w / v) sucrose.

[0065] Figure 20 The effect of 8.5% (w / v) sucrose in the freeze-dried hexavalent GBS vaccine at a dose of 50 mcg / ml (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on the antigenicity recovery of each serotype.

[0066] Figure 21 The effect of 7.0% (w / v) sucrose in the freeze-dried hexavalent GBS vaccine at a dose of 40 mcg / ml (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on the antigenicity recovery of each serotype.

[0067] Figure 22 The effect of 2.0% (w / v) sucrose and 4.0% (w / v) mannitol in the freeze-dried hexavalent GBS vaccine at a dose of 40 mcg / ml (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on the antigenicity recovery of each serotype.

[0068] Figure 23 The effect of 40 mcg / ml dose of freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) on the antigenicity recovery of each serotype with 3.0% (w / v) sucrose and 3.0% (w / v) mannitol.

[0069] Figure 24 The effect of 2.0% (w / v) sucrose and 4.0% (w / v) glycine in the freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) at a dose of 40 mcg / ml on the antigenicity recovery of each serotype.

[0070] Figure 25 The effect of 3.0% (w / v) sucrose and 3.0% (w / v) glycine in the freeze-dried hexavalent GBS vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 ) at a dose of 40 mcg / ml on the antigenicity recovery of each serotype. DETAILED DESCRIPTION OF THE INVENTION

[0071] It should be understood that the present invention is not limited to the specific methods and experimental conditions described, as these methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0072] Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, the preferred methods and materials are now described. The entire content of all publications mentioned herein is incorporated herein by reference.

[0073] The terms used herein have meanings as understood and known to those of ordinary skill in the art. However, for convenience and completeness, specific terms and their meanings are set forth below and throughout the specification.

[0074] Unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include plural referents. Thus, for example, reference to “the method” includes one or more methods and / or steps of the type described herein and / or those that will be apparent to one of ordinary skill in the art upon reading the present disclosure, and so forth.

[0075] The terms “about” or “approximately” mean within a statistically significant range of a numerical value. Such a range may be within an order of magnitude of the given numerical value or range, typically within 20%, more often within 10%, and even more often within 5%. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system being studied and can be readily understood by one of ordinary skill in the art. Whenever a range is recited in this application, each integer within that range is also considered to be an embodiment of the present invention.

[0076] It should be noted that, in the present disclosure, terms such as “comprises,” “comprised of,” “comprising,” “contains,” “containing,” etc. may have the meanings given to them in United States patent law; for example, they may mean “includes,” “included,” “including,” etc. Such terms refer to the inclusion of a specific component or group of components, but do not exclude any other components. Terms such as “consisting essentially of” have the meanings given to them in United States patent law; for example, they allow for the inclusion of additional components or steps that do not detract from the novelty or basic characteristics of the present invention, i.e., they exclude additional unrecited components or steps that would detract from the novelty or basic characteristics of the present invention, and they exclude components or steps of the prior art (such as the documents listed herein or incorporated by reference herein), especially those documents whose objective is to define patentability (e.g., novel, non-obvious, inventive compared to the prior art (e.g., compared to the documents listed or incorporated by reference herein)). And, the terms “consisting of” have the meanings given to them in United States patent law; that is, these terms are closed-ended. Thus, these terms refer to the inclusion of a specific component or group of components and exclude all other components.

[0077] The term "antigen" generally refers to a biomolecule that contains at least one epitope to which a homologous antibody can selectively bind, typically a protein, peptide, polysaccharide, lipid, or conjugate; or in some cases, "antigen" refers to an immunogenic substance that can stimulate an animal to produce antibodies or a T cell response, or both, including a composition that is injected or absorbed into an animal. An immune response can occur against the entire molecule or against one or more different parts of the molecule (such as an epitope or hapten). The term can be used to refer to an individual molecule or a homogeneous or heterogeneous population of antigen molecules. Antigens can be recognized by antibodies, T cell receptors, or other components of specific humoral and / or cellular immunity. The term "antigen" includes all relevant antigenic epitopes. The epitopes of a given antigen can be identified using any number of epitope mapping techniques well known in the art (see, for example, 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 by methods such as simultaneously synthesizing a large number of peptides on a solid support (such peptides corresponding to parts of the protein molecule) and reacting such 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, H.M., et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1984); Geysen, H.M., et al., Molec. Immunol., 23(7):709-715 (1986), the entire contents of which are incorporated herein by reference. Similarly, conformational epitopes can be identified by determining the spatial conformation of the amino acids, such as by, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols, supra). In addition, for the purposes of the present invention, "antigen" can also be used to refer to proteins that include modifications to the native sequence, such as deletions, insertions, and substitutions (which are generally conservative in nature, but can be non-conservative), provided that the protein retains the ability to elicit an immune response. These modifications can be deliberate, such as by site-directed mutagenesis, or by specific synthetic procedures, or by genetic engineering methods, or can be accidental, such as by mutations in the host that produces the antigen. In addition, antigens can be derived from, obtained from, or isolated from microorganisms (such as bacteria), or can be whole organisms. Similarly, oligonucleotides or polynucleotides that express antigens (such as in nucleic acid immunization applications) are also included in this definition.It also includes synthetic antigens, such as polyepitopes, flanking epitopes and other recombinant or synthetically derived antigens (Bergmann, C., et al., Eur. J. Immunol., 23(11):2777-2781(1993); Bergmann, C.C., et al., J. Immunol., 157(8):3242-3249(1996); Suhrbier, A., Immunol. and Cell Biol., 75(4):402-408(1997)).

[0078] The term "vaccine" or "vaccine composition" (which may be used interchangeably) refers to a pharmaceutical composition comprising at least one immunogenic composition capable of inducing an immune response in an animal.

[0079] Capsular polysaccharide

[0080] As used herein, the term "sugar" refers to a monosaccharide moiety or unit, and a combination of two or more monosaccharide 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.

[0081] As used herein, "monosaccharide" refers to a monosaccharide residue in an oligosaccharide. As used herein, the term "disaccharide" refers to a polysaccharide composed of two monosaccharide units or moieties linked together by a glycosidic bond.

[0082] In one embodiment, the polysaccharide is an oligosaccharide (OS). As used herein, "oligosaccharide" refers to a compound containing two or more monosaccharide units or moieties. In the context of oligosaccharides, the individual monomer units or moieties are monosaccharides, which are (or can be) bound to another monosaccharide unit or moiety through a hydroxyl group. Oligosaccharides can be prepared by chemical synthesis from protected single-residue sugars or by chemical degradation of biomanufactured polysaccharides. Alternatively, oligosaccharides can be manufactured by in vitro enzymatic methods.

[0083] In a preferred embodiment, the polysaccharide is a polysaccharide (PS), which refers to a linear or branched polymer having at least 5 monosaccharide units or moieties. For clarity, a larger number of repeating units (where n is greater than about 5, such as greater than about 10) will be referred to as a polysaccharide herein.

[0084] In one embodiment, the polysaccharide is a cell surface polysaccharide. Cell surface polysaccharide refers to a polysaccharide at least part of which is located on the outermost bacterial cell membrane or bacterial cell surface (including the peptidoglycan layer, cell wall and capsule). Generally, cell surface polysaccharides are associated with inducing an immune response in vivo. Cell surface polysaccharides can be "cell wall polysaccharides" or "capsular polysaccharides". Cell wall polysaccharides usually form a discontinuous layer on the bacterial surface.

[0085] In one embodiment, the polysaccharide is a capsular polysaccharide. A capsular polysaccharide refers to a sugar polymer comprising one or more repeating units of monosaccharides linked by glycosidic bonds. Capsular polysaccharides typically form a capsule-like layer around bacterial cells. "Capsular polysaccharide" refers to the polysaccharide capsule outside the cell wall of most Streptococcus isolates. For example, all GBS capsular polysaccharides have a branched repeating structure with terminal α2-3-linked sialic acid required for bacterial virulence. >94% of the invasive neonatal isolates from in vitro cultured T.E.S.T. were detected with capsular-associated sialic acid (quantified by HPLC analysis).

[0086] The inventors of the present application have found that the sialic acid level of GBS capsular polysaccharide is an important feature for generating an immune response. The prior art only provides conflicting information regarding the sialic acid level of serotype V, finding that desialylated serotype V is preferred (International Patent Application Publication No. WO 2012 / 035519) and serotype V with a sialic acid content >50% can be used (International Patent Application Publication No. WO 2014 / 053612). However, none of these references describe the importance of sialic acid level for the immunogenicity of at least most GBS polysaccharides. The inventors of the present application unexpectedly found that GBS capsular polysaccharide requires about 60% or more sialic acid before conjugation to provide an immune response comparable to those polysaccharides with natural sialic acid levels (i.e., 100% or more than about 95%). Even a sialic acid level of 58% (which is within the range of previously disclosed serotype V) still negatively affects immunogenicity.

[0087] Thus, in one embodiment of the present invention, the capsular polysaccharide comprises its natural sialic acid level (such as about 100% or more than about 95%). In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (sialylation degree higher than about 60%), such as up to about 35% (sialylation degree higher than about 65%), up to about 30% (sialylation degree higher than about 70%), up to about 25% (sialylation degree higher than about 75%), up to about 20% (sialylation degree higher than about 80%), up to about 15% (sialylation degree higher than about 85%), up to about 10% (sialylation degree higher than about 90%) and up to about 5% (sialylation degree higher than about 95%).

[0088] It should be noted that a 100% sialic acid level is equivalent to approximately 1.0 mM of sialic acid per mM of polysaccharide. Thus, the capsular polysaccharide can have approximately 1.0 mM of sialic acid per mM of polysaccharide, such as at least approximately 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least approximately 0.6 mM of sialic acid per mM of polysaccharide, such as at least approximately 0.65 mM of sialic acid per mM of polysaccharide, at least approximately 0.7 mM of sialic acid per mM of polysaccharide, at least approximately 0.75 mM of sialic acid per mM of polysaccharide, at least approximately 0.8 mM of sialic acid per mM of polysaccharide, at least approximately 0.85 mM of sialic acid per mM of polysaccharide, at least approximately 0.9 mM of sialic acid per mM of polysaccharide, or at least approximately 0.95 mM of sialic acid per mM of polysaccharide.

[0089] The terminal sialic acid residues of certain capsular polysaccharide (CP) serotypes are partially O-acetylated (OAc) (Lewis, A. L., 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 ∼40%), while serotypes Ia, II, and VII have little or no O-acetylation (less than approximately 5%) (Lewis 2004). In one embodiment of the invention, the capsular polysaccharide comprises its native level of O-acetylation (from about 0% to about 40%). In another embodiment, the capsular polysaccharide can be de-O-acetylated (less than about 5%). The degree of O-acetylation of the polysaccharide or oligosaccharide can be determined by any method known in the art, such as 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. WO 2005 / 033148 and WO 00 / 56357). Another commonly used method is that described by Hestrin, S., J. Biol. Chem., 180:249-261 (1949).

[0090] It should also be noted that 100% O-acetate is equivalent to approximately 1.0 mM O-acetate per mM of sugar repeat unit. Thus, a partially O-acetylated polysaccharide includes at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM of O-acetate per mM of sugar repeat unit. A de-O-acetylated polysaccharide includes less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM of O-acetate per mM of sugar repeat unit.

[0091] Streptococcal microorganisms capable of causing invasive disease typically also produce a CP that coats the bacterium and enhances its resistance to clearance by the host innate immune system. The CP serves to shield the bacterial cell within a protective capsule that renders the bacterium resistant to phagocytosis and intracellular killing. Bacteria lacking a capsule are more susceptible to phagocytosis. Capsular polysaccharides are often important virulence factors for many bacterial pathogens, including Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Staphylococcus aureus.

[0092] Capsular polysaccharides can be used to serotype specific bacterial species. Serotyping is typically accomplished by reacting with specific antisera or monoclonal antibodies raised against specific structures or unique epitope features of the capsular polysaccharide. There are ten GBS serotypes: Ia, Ib, and II through IX (Ferrieri, P., et al., Emerg. Infect. Dis. [Internet], 19(4) (2013), which is available at http: / / wwwnc.cdc.gov / eid / article / 19 / 4 / 12-1572_article).

[0093] In one embodiment of the present invention, the polysaccharide is isolated from Streptococcus agalactiae. The polysaccharide can be isolated from any encapsulated strain of S. agalactiae, such as 090, A909 (ATCC accession number BAA-1138), 515 (ATCC accession number BAA-1177), B523, CJB524, MB 4052 (ATCC accession number 31574), H36B (ATCC accession number 12401), S40, S42, MB 4053 (ATCC accession number 31575), M709, 133, 7357, PFEGBST0267, MB 4055 (ATCC accession number 31576), 18RS21 (ATCC accession number BAA-1175), S16, S20, V8 (ATCC accession number 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB 4082 (ATCC accession number 31577), M132, 110, M781 (ATCC accession number: BAA-22), D136C(3) (ATCC accession number 12403), M782, S23, 120, MB 4316 (M-732; ATCC accession number 31475), M132, K79, COH1 (ATCC accession number BAA-1176), PFEGBST0563, 3139 (ATCC accession number 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession number BAA-23), CJB112, 2603V / R (ATCC accession number BAA-611), NCTC 10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B 4589, B 4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62 and IT-PW-64.

[0094] The polysaccharides described herein can be isolated by methods known in the art, including, for example, the methods described herein. As used herein, "isolated" means obtained from and separated from a particular source. The term "isolated" further means not in its natural form, state, and / or environment. For example, "isolated from Streptococcus" means a substance obtained from and separated from Streptococcus cells. An isolated polysaccharide is not naturally occurring. The term "isolated" means that the substance has been removed from its original environment (e.g., from its natural environment if it is naturally occurring, or from its host organism if it is a recombinant entity, or moved 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 when it is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, or otherwise present as part of a chemical reaction in a mixture. In the present invention, a protein or polysaccharide can be isolated from bacterial cells or from cell debris so as to be provided in a form useful for making an immunogenic composition. The term "isolated" or "isolation" can include purification, including methods known in the art for purifying isolated polysaccharides and / or the methods described herein. "Substantially free of cellular material" includes polypeptide / protein preparations in which the polypeptide / protein is separated from the 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 polysaccharide, protein, or peptide having less than about 30%, 20%, 10%, 5%, 2.5%, or 1% (by dry weight) of contaminating protein or polysaccharide or other cellular material. When the polypeptide / protein is recombinantly produced, it is also preferably substantially free of the culture medium, i.e., the protein preparation includes less than about 20%, 10%, or 5% by volume of the culture medium. When the polypeptide / protein or polysaccharide is produced by chemical synthesis, preferably, it is substantially free of chemical precursors or other chemicals, i.e., it is separated from the chemical precursors or other chemicals involved in the synthesis of the protein or polysaccharide. Thus, a preparation of such a polypeptide / protein or polysaccharide has less than about 30%, 20%, 10%, 5% (by dry weight) of non-target polypeptide / protein or polysaccharide fragments of chemical precursors or compounds.

[0095] In one embodiment of the present invention, the polysaccharide is isolated from bacteria. In another embodiment of the present invention, the polysaccharide is produced recombinantly. In another embodiment, the polysaccharide is synthesized according to conventional methods or chemically synthesized. In another embodiment of the present invention, the polysaccharide is produced by cloning and expressing the biosynthetic pathway that produces the polysaccharide and then expressing it in an alternative host. In one embodiment, the polysaccharide is immunogenic. For example, the inventors of the present application have found that each of the polysaccharides described herein is capable of inducing or eliciting an immune response. The term "immunogenic" refers to the ability to initiate, trigger, cause, enhance, improve, and / or augment 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.

[0096] The molecular weight of the capsular polysaccharide is a consideration for immunogenic compositions. High molecular weight capsular polysaccharides are capable of inducing certain antibody immune responses due to the higher valence of epitopes present on the antigen surface. The isolation and purification of high molecular weight capsular polysaccharides are contemplated for the conjugates, compositions, and methods of the present invention.

[0097] However, in one embodiment, the size of the polysaccharide can be altered to a molecular weight (MW) range that is less than the molecular weight of the native capsular polysaccharide (before conjugation to a carrier protein). The size of the purified capsular polysaccharide is reduced to produce conjugates having favorable filtration characteristics and / or yield.

[0098] In one such embodiment, the size of the purified capsular polysaccharide is reduced by high pressure homogenization. High pressure homogenization achieves high shear rates by pumping the process stream through a flow path that is small enough. The shear rate is increased by using a larger applied homogenization pressure, and the exposure time can be increased by recycling the feed stream through the homogenizer.

[0099] In one embodiment, the polysaccharides described herein are capable of inducing opsonin activity. In another embodiment, the polysaccharides described herein are capable of inducing opsonin activity and phagocytic activity (e.g., opsonophagocytic activity).

[0100] Opsonin activity or opsonization refers to the process by which opsonins (e.g., antibodies or complement factors) bind to an antigen (e.g., the isolated polysaccharides described herein), which promotes the attachment of the antigen to phagocytic cells or phagocytes (e.g., macrophages, dendritic cells, and polymorphonuclear leukocytes (PMNL)). Certain bacteria (such as, for example, encapsulated bacteria that are not normally phagocytosed due to the presence of a capsule) can become more readily recognized by phagocytes when coated with opsonin antibodies. In one embodiment, the polysaccharide induces an immune response, such as, for example, an antibody, that is an opsonin. In one embodiment, the opsonin activity is directed against Gram-positive cocci, preferably against Streptococcus species, and more preferably against at least one strain of Streptococcus agalactiae.

[0101] In another embodiment, the polysaccharides described herein are capable of inducing a bactericidal immune response. In one embodiment, the bactericidal activity is directed against Gram-positive cocci, preferably against Streptococcus species, more preferably against at least one strain of Streptococcus agalactiae.

[0102] Methods for measuring opsonization, phagocytosis, and / or bactericidal activity are known in the art and can be carried out, for example, by measuring the reduction of bacterial load in vivo (e.g., by measuring the level of bacteremia in a mammal challenged with Streptococcus species) and / or by measuring bacterial cell killing in vitro (e.g., an in vitro opsonophagocytic assay). In one embodiment, the polysaccharide is capable of inducing opsonin, phagocytic, and / or bactericidal activity when compared to an appropriate control group (such as, for example, when compared to an antiserum generated against heat-killed Gram-positive cocci).

[0103] Serotype Ia

[0104] One embodiment includes the serotype Ia GBS capsular polysaccharide. The structure of serotype Ia can be described as follows:

[0105]

[0106] The molecular weight of the serotype Ia capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 kDa to about 400 kDa, from about 25 kDa to about 350 kDa, from about 25 kDa to about 300 kDa, from about 25 kDa to about 250 kDa, from about 25 kDa to about 200 kDa, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In a preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 25 kDa to about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 100 kDa to about 400 kDa.Any integer within any of the above ranges is considered as an embodiment of the present disclosure.

[0107] In certain embodiments, a high-pressure homogenization process is used to reduce the size of native GBS capsular polysaccharide serotype Ia while preserving the structural features of the polysaccharide, such as sialic acid.

[0108] In one embodiment of the invention, the serotype Ia capsular polysaccharide contains its native sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (sialylation level higher than about 60%) before conjugation, such as up to about 35% (sialylation level higher than about 65%), up to about 30% (sialylation level higher than about 70%), up to about 25% (sialylation level higher than about 75%), up to about 20% (sialylation level higher than about 80%), up to about 15% (sialylation level higher than about 85%), up to about 10% (sialylation level higher than about 90%), or up to about 5% (sialylation level higher than about 95%).

[0109] In another embodiment, the serotype Ia capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0110] The O - acetylation of the serotype Ia capsular polysaccharide is less than about 5%. Some exemplary strains of the serotype Ia capsular polysaccharide of the present invention include 090, A909 (ATCC accession number BAA - 1138), 515 (ATCC accession number BAA - 1177), B523, CJB524, and MB 4052 (ATCC accession number 31574).

[0111] Serotype Ib

[0112] One embodiment includes the serotype Ib GBS capsular polysaccharide. The structure of serotype Ib can be described as follows:

[0113]

[0114] The molecular weight of the serotype Ib capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 kDa to about 400 kDa, from about 25 kDa to about 350 kDa, from about 25 kDa to about 300 kDa, from about 25 kDa to about 250 kDa, from about 25 kDa to about 200 kDa, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In a preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 25 kDa to about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0115] In one embodiment of the present invention, the serotype Ib capsular polysaccharide contains its natural sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (sialylation degree higher than about 60%) before conjugation, such as up to about 35% (sialylation degree higher than about 65%), up to about 30% (sialylation degree higher than about 70%), up to about 25% (sialylation degree higher than about 75%), up to about 20% (sialylation degree higher than about 80%), up to about 15% (sialylation degree higher than about 85%), up to about 10% (sialylation degree higher than about 90%), or up to about 5% (sialylation degree higher than about 95%).

[0116] In another embodiment, the serotype Ib capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0117] The serotype Ib capsular polysaccharide is about 0% to about 40% O-acetylated. In one embodiment of the present invention, the polysaccharide is de-O-acetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of the serotype Ib capsular polysaccharide of the present invention include H36B (ATCC accession number 12401), S40, S42, MB 4053 (ATCC accession number 31575), M709, 133, 7357, and PFEGBST0267.

[0118] Serotype II

[0119] One embodiment includes the serotype II GBS capsular polysaccharide. The structure of serotype II can be described as follows:

[0120]

[0121] The molecular weight of the serotype II capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 kDa to about 400 kDa, from about 25 kDa to about 350 kDa, from about 25 kDa to about 300 kDa, from about 25 kDa to about 250 kDa, from about 25 kDa to about 200 kDa, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In a preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 25 kDa to about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0122] In one embodiment of the present invention, the serotype II capsular polysaccharide contains its natural sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (sialylation degree higher than about 60%) before conjugation, such as up to about 35% (sialylation degree higher than about 65%), up to about 30% (sialylation degree higher than about 70%), up to about 25% (sialylation degree higher than about 75%), up to about 20% (sialylation degree higher than about 80%), up to about 15% (sialylation degree higher than about 85%), up to about 10% (sialylation degree higher than about 90%), or up to about 5% (sialylation degree higher than about 95%).

[0123] In another embodiment, the serotype II capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0124] The O - acetylation of the serotype II capsular polysaccharide is less than about 5%. Some exemplary strains of the serotype II capsular polysaccharide of the present invention include MB 4055 (ATCC accession number 31576), 18RS21 (ATCC accession number BAA - 1175), S16, S20, V8 (ATCC accession number 12973), DK21, DK23, UAB, 5401, and PFEGBST0708.

[0125] Serotype III

[0126] One embodiment includes the serotype III GBS capsular polysaccharide. The structure of serotype III can be described as follows:

[0127]

[0128] The molecular weight of the serotype III capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 kDa to about 400 kDa, from about 25 kDa to about 350 kDa, from about 25 kDa to about 300 kDa, from about 25 kDa to about 250 kDa, from about 25 kDa to about 200 kDa, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In a preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 25 kDa to about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 100 kDa to about 400 kDa.Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0129] In certain embodiments, the size of native GBS capsular polysaccharide serotype III is reduced using a high-pressure homogenization process while preserving the structural features of the polysaccharide, such as sialic acid.

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

[0131] In another embodiment, the serotype III capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0132] The serotype III capsular polysaccharide is about 0% to about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is de-O-acetylated (i.e., O-acetylation less than about 5%). Some exemplary strains of the serotype III capsular polysaccharide of the present invention include MB 4082 (ATCC accession number 31577), M132, 110, M781 (ATCC accession number BAA-22), D136C(3) (ATCC accession number 12403), M782, S23, 120, MB 4316 (M-732; ATCC accession number 31475), M132, K79, COH1 (ATCC accession number BAA-1176), and PFEGBST0563.

[0133] Serotype IV

[0134] One embodiment includes a serotype IV GBS capsular polysaccharide. The structure of serotype IV can be described as follows:

[0135]

[0136] The molecular weight of the serotype IV capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 kDa to about 400 kDa, from about 25 kDa to about 350 kDa, from about 25 kDa to about 300 kDa, from about 25 kDa to about 250 kDa, from about 25 kDa to about 200 kDa, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In a preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 25 kDa to about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0137] In one embodiment of the present invention, the serotype IV capsular polysaccharide contains its natural sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (degree of sialylation higher than about 60%) before conjugation, such as up to about 35% (degree of sialylation higher than about 65%), up to about 30% (degree of sialylation higher than about 70%), up to about 25% (degree of sialylation higher than about 75%), up to about 20% (degree of sialylation higher than about 80%), up to about 15% (degree of sialylation higher than about 85%), up to about 10% (degree of sialylation higher than about 90%), or up to about 5% (degree of sialylation higher than about 95%).

[0138] In another embodiment, the serotype IV capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0139] The serotype IV capsular polysaccharide is about 0% to about 40% O - acetylated. In one embodiment of the present invention, the polysaccharide is de - O - acetylated (i.e., O - acetylation less than about 5%). Some exemplary strains of the serotype IV capsular polysaccharide of the present invention include 3139 (ATCC accession number 49446), CZ - NI - 016, and PFEGBST0961.

[0140] Serotype V

[0141] One embodiment includes the serotype V GBS capsular polysaccharide. The structure of serotype V can be described as follows:

[0142]

[0143] The molecular weight of the serotype V capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 kDa to about 400 kDa, from about 25 kDa to about 350 kDa, from about 25 kDa to about 300 kDa, from about 25 kDa to about 250 kDa, from about 25 kDa to about 200 kDa, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In a preferred embodiment, the molecular weight of the capsular polysaccharide before conjugation is from about 25 kDa to about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0144] In one embodiment of the present invention, the serotype V capsular polysaccharide contains its native sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (degree of sialylation higher than about 60%) before conjugation, such as up to about 35% (degree of sialylation higher than about 65%), up to about 30% (degree of sialylation higher than about 70%), up to about 25% (degree of sialylation higher than about 75%), up to about 20% (degree of sialylation higher than about 80%), up to about 15% (degree of sialylation higher than about 85%), up to about 10% (degree of sialylation higher than about 90%), or up to about 5% (degree of sialylation higher than about 95%).

[0145] In another embodiment, the serotype V capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0146] The serotype V capsular polysaccharide is about 0% to about 40% O-acetylated. In one embodiment of the present invention, the polysaccharide is de-O-acetylated (i.e., O-acetylation less than about 5%). Some exemplary strains of the serotype V capsular polysaccharide of the present invention include 1169-NT1, CJB111 (ATCC accession number BAA-23), CJB112, 2603V / R (ATCC accession number BAA-611), NCTC10 / 81, CJ11, and PFEGBST0837.

[0147] Serotype VI

[0148] The GBS serotype VI capsular polysaccharide is described by von Hunolstein, C., et al., Infection and Immunity, 61(94):1272-1280 (1993) (the entire disclosure of which is incorporated herein by reference). The structure of serotype VI can be described as follows:

[0149]

[0150] The molecular weight of the serotype VI capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0151] In one embodiment of the present invention, the serotype VI capsular polysaccharide comprises its natural sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (sialylation degree higher than about 60%) before conjugation, such as up to about 35% (sialylation degree higher than about 65%), up to about 30% (sialylation degree higher than about 70%), up to about 25% (sialylation degree higher than about 75%), up to about 20% (sialylation degree higher than about 80%), up to about 15% (sialylation degree higher than about 85%), up to about 10% (sialylation degree higher than about 90%), or up to about 5% (sialylation degree higher than about 95%).

[0152] In another embodiment, the serotype VI capsular polysaccharide has about 1.0 mM sialic acid per mM of polysaccharide, such as at least about 0.95 mM sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM sialic acid per mM of polysaccharide, at least about 0.7 mM sialic acid per mM of polysaccharide, at least about 0.75 mM sialic acid per mM of polysaccharide, at least about 0.8 mM sialic acid per mM of polysaccharide, at least about 0.85 mM sialic acid per mM of polysaccharide, at least about 0.9 mM sialic acid per mM of polysaccharide, or at least about 0.95 mM sialic acid per mM of polysaccharide.

[0153] The serotype VI capsular polysaccharide is about 0% to about 40% O-acetylated. In one embodiment of the present invention, the polysaccharide is de-O-acetylated (i.e., O-acetylation is less than about 5%). Some exemplary strains of the serotype VI capsular polysaccharide of the present invention include 118754, 114852, 114862, 114866, 118775, B 4589, B 4645, SS1214, and CZ-PW-119.

[0154] Serotype VII

[0155] The GBS serotype VII capsular polysaccharide is described by Kogan, G., et al., Carbohydrate Research, 277(1):1-9(1995) (the entire disclosure of which is incorporated herein by reference). The repeating unit of serotype VII is as follows:

[0156]

[0157] The molecular weight of the serotype VII capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0158] In one embodiment of the present invention, the serotype VII capsular polysaccharide comprises its native sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (sialylation degree higher than about 60%) before conjugation, such as up to about 35% (sialylation degree higher than about 65%), up to about 30% (sialylation degree higher than about 70%), up to about 25% (sialylation degree higher than about 75%), up to about 20% (sialylation degree higher than about 80%), up to about 15% (sialylation degree higher than about 85%), up to about 10% (sialylation degree higher than about 90%), or up to about 5% (sialylation degree higher than about 95%).

[0159] In another embodiment, the serotype VII capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0160] The O - acetylation of the serotype VII capsular polysaccharide is less than about 5%. Some exemplary strains of the serotype VII capsular polysaccharide of the present invention include 7271 and CZ - PW - 045.

[0161] Serotype VIII

[0162] The GBS serotype VIII capsular polysaccharide is described by Kogan, G., et al., The Journal of Biological Chemistry, 271(15):8786 - 8790(1996) (the entire disclosure of which is incorporated herein by reference). The repeating unit of serotype VIII is as follows:

[0163]

[0164] The molecular weight of the serotype VIII capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0165] In one embodiment of the present invention, the serotype VIII capsular polysaccharide contains its natural sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% before conjugation (degree of sialylation higher than about 60%), such as up to about 35% (degree of sialylation higher than about 65%), up to about 30% (degree of sialylation higher than about 70%), up to about 25% (degree of sialylation higher than about 75%), up to about 20% (degree of sialylation higher than about 80%), up to about 15% (degree of sialylation higher than about 85%), up to about 10% (degree of sialylation higher than about 90%), or up to about 5% (degree of sialylation higher than about 95%).

[0166] In another embodiment, the serotype VIII capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0167] The serotype VIII capsular polysaccharide is about 0% to about 40% O-acetylated. In one embodiment of the present invention, the polysaccharide is de-O-acetylated (i.e., O-acetylation less than about 5%). Some exemplary strains of the serotype VIII capsular polysaccharide of the present invention include JM9130013 and JM9130672.

[0168] Serotype IX

[0169] The GBS serotype IX capsular polysaccharide is described by Berti, F., et al., The Journal of Biological Chemistry, 289(34):23437-2348(2014) (the entire disclosure of which is incorporated herein by reference). The structure of serotype IX can be described as follows:

[0170]

[0171] The molecular weight of the serotype IX capsular polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0172] In one embodiment of the present invention, the serotype IX capsular polysaccharide contains its native sialic acid level, such as about 100% or higher than about 95%. In another embodiment, the capsular polysaccharide can be desialylated up to about 40% (sialylation level higher than about 60%) before conjugation, such as up to about 35% (sialylation level higher than about 65%), up to about 30% (sialylation level higher than about 70%), up to about 25% (sialylation level higher than about 75%), up to about 20% (sialylation level higher than about 80%), up to about 15% (sialylation level higher than about 85%), up to about 10% (sialylation level higher than about 90%), or up to about 5% (sialylation level higher than about 95%).

[0173] In another embodiment, the serotype IX capsular polysaccharide has about 1.0 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.95 mM of sialic acid per mM of polysaccharide. In another embodiment, the capsular polysaccharide can have at least about 0.6 mM of sialic acid per mM of polysaccharide before conjugation, such as at least about 0.65 mM of sialic acid per mM of polysaccharide, at least about 0.7 mM of sialic acid per mM of polysaccharide, at least about 0.75 mM of sialic acid per mM of polysaccharide, at least about 0.8 mM of sialic acid per mM of polysaccharide, at least about 0.85 mM of sialic acid per mM of polysaccharide, at least about 0.9 mM of sialic acid per mM of polysaccharide, or at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0174] The serotype IX capsular polysaccharide is O-acetylated from about 0% to about 40%. In one embodiment of the present invention, the polysaccharide is de-O-acetylated (i.e., O-acetylation less than about 5%). Some exemplary strains of the serotype IX capsular polysaccharide of the present invention include IT-NI-016, IT-PW-62, and IT-PW-64.

[0175] Polysaccharide-protein conjugate

[0176] As used herein, "conjugate" includes a capsular polysaccharide (usually having a desired molecular weight range) and a carrier protein, wherein 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 a capsular polysaccharide that is non-covalently associated (i.e., not covalently bound to, adsorbed to, or entrapped within the conjugated capsular polysaccharide-carrier protein) with the conjugated capsular polysaccharide-carrier protein. The terms "free capsular polysaccharide", "free polysaccharide", and "free sugar" may be used interchangeably and are intended to convey the same meaning. Regardless of the nature of the carrier molecule, it may be conjugated directly or through a linker to the capsular polysaccharide. As used herein, "conjugation" refers to the process of covalently linking a bacterial capsular polysaccharide to a carrier molecule. Conjugation enhances the immunogenicity of the bacterial capsular polysaccharide. Conjugation can be carried out according to the methods described below or by processes known in the art.

[0177] As used herein, "conjugate immunogenic composition" refers to an immunogenic composition wherein the immunogenic substance includes an antigenic polysaccharide covalently linked to a carrier protein to produce a polysaccharide-protein conjugate. In one embodiment, the polysaccharide-protein conjugate of the present invention can be formulated into a multivalent immunogenic composition.

[0178] As used herein, the term "molecular weight" of a polysaccharide or a carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) in combination with a multi-angle laser light scattering detector (MALLS).

[0179] 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". Carrier proteins are used to enhance the antigenicity and immunogenicity of the polysaccharide. As used herein, the term "carrier effect" refers to the process by which the antigenicity and immunogenicity of a weakly immunogenic or non-immunogenic molecule are enhanced by linking it to a more immunogenic molecule (such as a heterologous protein) serving as a carrier. In this case, the polysaccharide in the combined polysaccharide-protein conjugate becomes more immunogenic than when it exists alone. Carrier proteins contain T cell epitopes for stimulating T cells to assist in the production of an antibody response.

[0180] As used herein, "carrier protein" or "protein carrier" refers to any protein molecule that can be conjugated to an antigen (such as a capsular polysaccharide) corresponding to the desired immune response. Conjugation of an antigen (such as a polysaccharide) to a carrier protein can render the antigen immunogenic. The carrier protein is preferably a protein that is non-toxic and non-reactive and is available in sufficient quantity and purity. Examples of carrier proteins are toxins, toxoids, or any mutant cross-reacting material (CRM 197 ) from tetanus, diphtheria, pertussis, species of Pseudomonas, E. coli, species of Staphylococcus, and species of Streptococcus. The carrier protein should be able to withstand standard conjugation procedures. In a particular embodiment of the present invention, CRM 197 is used as the carrier protein.

[0181] Cross-reacting material or CRM is particularly useful in some embodiments of the present invention. Genetically altered 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 CRM. CRM 197 (Wyeth / Pfizer Inc., Sanford, NC) is notable because it has a single amino acid change from native diphtheria toxin and is immunologically indistinguishable from it. See Pappenheimer, A.M., et al., Immunochem., 9(9):891-906 (1972), U.S. Patent No. 5,614,382, the entire disclosure of which is incorporated herein by reference. CRM 197 is a non-toxic variant of diphtheria toxin (i.e., a toxoid) that is isolated from a culture of Corynebacterium diphtheriae strain C7(β197) grown in a basal medium of casein amino acids and yeast extract. CRM 197 is purified by ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. The culture of Corynebacterium diphtheriae strain C7 (C. diphtheriae strain C7)(β197) that produces the CRM 197 protein has been deposited with the American Type Culture Collection (Rockville, Maryland) and has been assigned 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, W.J., et al., Science, 240(4852):656-659 (1988), the entire disclosure of which is incorporated herein by reference.

[0182] In addition to diphtheria toxoid (DT), CRM 197 and pertussis toxoid, other examples of carrier proteins include tetanus toxoid (TT), cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004 / 083251), Escherichia coli heat-labile enterotoxin (LT), Escherichia coli heat-stable enterotoxin (ST), pneumolysin from Streptococcus pneumoniae (wild type or mutant with reduced toxicity), 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 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 of tuberculin (PPD), 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 mixture c (OMPC), porin, transferrin-binding protein, or Clostridium difficile enterotoxins (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 CRM 197 . In another embodiment of the present invention, the carrier protein is tetanus toxoid.

[0183] To synthesize a multivalent conjugate immunogenic composition, the polysaccharide-protein conjugate can be produced by conjugating a mixture of polysaccharides purified from two different bacterial species to a carrier protein. Alternatively, a multivalent conjugate immunogenic composition can be manufactured by combining polysaccharides purified from two or more different serotypes of the same bacterium and conjugating them in mixture form to 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, the multivalent immunogenic composition can include a homogeneous or heterogeneous population of carrier proteins with attached polysaccharides.

[0184] After conjugating the capsular polysaccharide with a carrier protein, the polysaccharide-protein conjugate (enriched in terms of the amount of the polysaccharide-protein conjugate) is purified by various techniques. These techniques include, for example, concentration / diafiltration operations, precipitation / elution, column chromatography, and depth filtration.

[0185] As described above, the present invention relates to a conjugate comprising a GBS capsular polysaccharide conjugated with a carrier protein. One embodiment of the present invention provides a conjugate comprising a GBS serotype IV capsular polysaccharide conjugated with a carrier protein and at least one additional conjugate, the additional conjugate comprising a GBS serotype Ia capsular polysaccharide conjugated with a carrier protein, a GBS serotype Ib capsular polysaccharide conjugated with a carrier protein, a GBS serotype II capsular polysaccharide conjugated with a carrier protein, a GBS serotype III capsular polysaccharide conjugated with a carrier protein, a GBS serotype V capsular polysaccharide conjugated with a carrier protein, a GBS serotype VI capsular polysaccharide conjugated with a carrier protein, a GBS serotype VII capsular polysaccharide conjugated with a carrier protein, a GBS serotype VIII capsular polysaccharide conjugated with a carrier protein, or a GBS serotype IX capsular polysaccharide conjugated with a carrier protein. In one aspect of the present invention, the molecular weight of the polysaccharide is from about 5 kDa to 1,000 kDa; the molecular weight of the conjugate is from about 300 kDa to about 20,000 kDa; and the conjugate comprises less than about 40% free polysaccharide compared to the total amount of the polysaccharide. In 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 compared to the total amount of the polysaccharide.

[0186] In one embodiment, the molecular weight of serotype Ia, Ib, II, III, IV, V, VI, VII, VIII, and / or IX polysaccharide before conjugation is from about 5 kDa to about 1,000 kDa, such as from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0187] In one embodiment, the conjugate has a molecular weight of from about 300 kDa to about 20,000 kDa, such as from about 300 kDa to about 15,000 kDa, from about 300 kDa to about 10,000 kDa, from about 300 kDa to about 9,000 kDa, from about 300 kDa to about 8,000 kDa, from about 300 kDa to about 7,000 kDa, from about 300 kDa to about 6,000 kDa, from about 300 kDa to about 5,000 kDa, from about 300 kDa to about 4,000 kDa, from about 300 kDa to about 3,000 kDa, from about 300 kDa to about 2,000 kDa, from about 300 kDa to about 1,000 kDa, from about 500 kDa to about 20,000 kDa, from about 500 kDa to about 15,000 kDa, from about 500 kDa to about 10,000 kDa, from about 500 kDa to about 9,000 kDa, from about 500 kDa to about 8,000 kDa, from about 500 kDa to about 7,000 kDa, from about 500 kDa to about 6,000 kDa, from about 500 kDa to about 5,000 kDa, from about 500 kDa to about 4000 kDa, from about 500 kDa to about 3,000 kDa, from about 500 kDa to about 2,000 kDa, from about 500 kDa to about 1,000 kDa, from about 1,000 kDa to about 20,000 kDa, from about 1,000 kDa to about 15,000 kDa, from about 1,000 kDa to about 10,000 kDa, from about 1,000 kDa to about 9,000 kDa, from about 1,000 kDa to about 8,000 kDa, from about 1,000 kDa to about 7,000 kDa, from about 1,000 kDa to about 6,000 kDa, from about 1,000 kDa to about 5,000 kDa, from about 1,500 kDa to about 20,000 kDa, from about 1,500 kDa to about 15,000 kDa, from about 1,500 kDa to about 10,000 kDa, from about 1,500 kDa to about 9,000 kDa, from about 1,500 kDa to about 8,000 kDa, from about 1,500 kDa to about 7,000 kDa, from about 1,500 kDa to about 6,000 kDa, from 1500 kDa to about 5,000 kDa, from about 2,000 kDa to about 20,000 kDa, from about 2,000 kDa to about 15,000 kDa, from about 2,000 kDa to about 10,000 kDa, from about 2,000 kDa to about 9,000 kDa, from about 2,000 kDa to about 8,000 kDa, from about 2,000 kDa to about 7,000 kDa, from about 2,000 kDa to about 6,000 kDa, from about 2,500 kDa to about 20,000 kDa, from about 2,500 kDa to about 15,000 kDa, from about 2,500 kDa to about 10,000 kDa, from about 2,500 kDa to about 9,about 0 kDa, from about 2,500 kDa to about 8,000 kDa, from about 2,500 kDa to about 7,000 kDa, from about 2,500 kDa to about 6,000 kDa, from about 3,000 kDa to about 20,000 kDa, from about 3,000 kDa to about 15,000 kDa, from about 3,000 kDa to about 10,000 kDa, from about 3,000 kDa to about 9,000 kDa, from about 3,000 kDa to about 8,000 kDa, from about 3,000 kDa to about 7,000 kDa, or from about 3,000 kDa to about 6,000 kDa.,

[0188] In one embodiment, the GBS serotype IV capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0189] In one embodiment, the GBS serotype Ia capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0190] In one embodiment, the GBS serotype Ib capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0191] In one embodiment, the GBS serotype II capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0192] In one embodiment, the GBS serotype III capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0193] In one embodiment, the GBS serotype V capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0194] In one embodiment, the GBS serotype VI capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0195] In one embodiment, the GBS serotype VII capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0196] In one embodiment, the GBS serotype VIII capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0197] In one embodiment, the GBS serotype IX capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0198] In one embodiment, the conjugate of the present invention has 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 of sialic acid per mM of polysaccharide. In a preferred embodiment, the conjugate has at least about 0.9 or 0.95 mM of sialic acid per mM of polysaccharide.

[0199] In one embodiment, the GBS serotype IV capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0200] In one embodiment, the GBS serotype Ia capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0201] In one embodiment, the GBS serotype Ib capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0202] In one embodiment, the GBS serotype II capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0203] In one embodiment, the GBS serotype III capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0204] In one embodiment, the GBS serotype V capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0205] In one embodiment, the GBS serotype VI capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0206] In one embodiment, the GBS serotype VII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0207] In one embodiment, the GBS serotype VIII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0208] In one embodiment, the GBS serotype IX capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0209] In one embodiment, the conjugate of the present invention has less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM sugar repeat unit. In another embodiment, the conjugate contains at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM O-acetate per mM sugar repeat unit.

[0210] In one embodiment, the GBS serotype IV capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0211] In one embodiment, the GBS serotype Ia capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0212] In one embodiment, the GBS serotype Ib capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0213] In one embodiment, the GBS serotype II capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0214] In one embodiment, the GBS serotype III capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0215] In one embodiment, the GBS serotype V capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0216] In one embodiment, the GBS serotype VI capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0217] In one embodiment, the GBS serotype VII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0218] In one embodiment, the GBS serotype VIII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0219] In one embodiment, the GBS serotype IX capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0220] In another embodiment, compared to the total amount of GBS capsular polysaccharide, the immunogenic conjugate contains 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% of free GBS capsular polysaccharide. In a preferred embodiment, compared to the total amount of GBS capsular polysaccharide, the immunogenic conjugate contains less than about 5% of unreacted free sugar.

[0221] In another embodiment, the ratio (weight to weight) of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.5 to about 3.0. In one aspect, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.5 to about 2.0, from about 0.5 to about 1.5, from about 0.5 to about 1.0, from about 1.0 to about 1.5, or from about 1.0 to about 2.0. In a preferred embodiment, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.8 to about 1.0.

[0222] In another embodiment, the degree of conjugation of the conjugate is 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 15, 3 to 13, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 5 to 15, 5 to 10, 8 to 15, 8 to 12, 10 to 15, or 10 to 12. In a preferred embodiment, the degree of conjugation of the conjugate is 2 to 5.

[0223] Conjugation

[0224] Conjugation can be direct, where an atom from the polysaccharide is covalently bonded to an atom from the protein surface. Alternatively, conjugation can be through a linker molecule that reacts with both the polysaccharide and the protein and joins the two, thereby tethering the carbohydrate to the protein.

[0225] When the carrier is conjugated (i.e., covalently bound) to one or more antigens such as polysaccharides, the conjugation can be carried out by any chemical method, process, or genetic technique known in the art. For example, the carrier polypeptide can be conjugated to one or more antigens selected from the group consisting of 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 by a variety of techniques, including but not limited to: (1) direct coupling through protein functional groups (e.g., thiol - thiol linkage, amine - carboxyl linkage, amine - aldehyde linkage; direct enzyme coupling); (2) homobifunctional coupling of amines (e.g., using bis - aldehydes); (3) homobifunctional coupling of thiols (e.g., using bis - maleimides); (4) homobifunctional coupling through photoactivating reagents; (5) heterobifunctional coupling of amines and thiols (e.g., using maleimides); (6) heterobifunctional coupling through photoactivating reagents (e.g., β - carbonyl diazo groups); (7) introduction of amine - reactive groups into polysaccharides or oligosaccharides by cyanogen bromide activation or carboxymethylation; (8) introduction of thiol - reactive groups into polysaccharides or oligosaccharides by heterobifunctional compounds such as maleimidyl - hydrazide; (9) protein - lipid conjugation by introducing hydrophobic groups into the protein and (10) protein - lipid conjugation by incorporating reactive groups into the lipid. In addition, heterobifunctional "non - covalent coupling" techniques such as biotin - avidin interactions are contemplated. Other methods well - known in the art for conjugating oligosaccharides and polysaccharides to immunogenic carrier proteins are also within the scope of certain embodiments of the present invention.

[0226] 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 directly coupled to the amino groups on the carrier protein or through a spacer. For example, the spacer can be cystamine or cysteamine to produce a thiolated polysaccharide, which can be coupled to the carrier through a thioether linkage obtained after reaction with maleimide-activated carrier protein (e.g., using GMBS) or haloacetylated carrier protein (e.g., using iodoacetamide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP).

[0227] In one aspect, the cyanate ester (optionally chemically produced by CDAP) is coupled to hexamethylenediamine or adipic dihydrazide (ADH), and the amino-derivatized sugar is conjugated to the carrier protein through the carboxyl groups on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugates are described, for example, in International Patent Application Publication Nos. WO 93 / 15760, WO 95 / 08348, and WO 96 / 29094.

[0228] Other suitable techniques use carbodiimide, hydrazide, active ester, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, and TSTU. There are many descriptions in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl spacer, which can be formed by reacting the free hydroxyl groups 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 linkage. This may involve reducing the anomeric end to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI / CDT to form a CDI / CDT carbamate intermediate, and coupling the CDI / CDT carbamate intermediate to the amino groups on the protein.

[0229] In a preferred embodiment, the GBS capsular polysaccharide-protein conjugate of the present invention is manufactured using reductive amination. Reductive amination involves two steps: (1) oxidizing the polysaccharide to generate aldehyde functionality from adjacent diols in individual hexose units and (2) reducing the activated polysaccharide and the carrier protein to form the conjugate.

[0230] In one embodiment, the GBS capsular polysaccharide is activated (oxidized) by a method comprising the following steps:

[0231] (a) React the isolated GBS capsular polysaccharide with an oxidizing agent; and

[0232] (b) Quench the oxidation reaction by adding a quenching agent to produce the activated GBS capsular polysaccharide.

[0233] In one aspect of the present invention, the concentration of the isolated capsular polysaccharide is from about 0.1 mg / mL to about 10.0 mg / mL, such as from about 0.5 mg / mL to about 5.0 mg / mL, from about 1.0 mg / mL to about 3.0 mg / mL, or about 2.0 mg / mL.

[0234] In certain embodiments, the oxidizing agent is periodate. Periodate oxidizes adjacent hydroxyl groups to form carbonyl or aldehyde groups and causes C-C bond cleavage. The term "periodate" includes both periodate and periodic acid. The term also includes metaperiodate (IO 4 - ) and orthoperiodate (IO 6 5- ). The term "periodate" also includes various periodates, 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 the GBS capsular polysaccharide is metaperiodate. In a preferred embodiment, the periodate used to oxidize the serotype capsular polysaccharide is sodium metaperiodate.

[0235] 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 another embodiment, the polysaccharide is reacted with about 0.1 molar equivalent of the oxidizing agent. In another embodiment, the polysaccharide is reacted with about 0.15 molar equivalent of the oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.25 molar equivalent of the oxidizing agent. In another embodiment, the polysaccharide is reacted with about 0.5 molar equivalent of the oxidizing agent. In an alternative embodiment, the polysaccharide is reacted with about 0.6 molar equivalent of the oxidizing agent. In another embodiment, the polysaccharide is reacted with about 0.7 molar equivalent of the oxidizing agent.

[0236] In one aspect of the present invention, the duration of the oxidation reaction is about 1 hour to about 50 hours, about 10 hours to about 30 hours, about 15 hours to about 20 hours, about 15 hours to about 17 hours, or about 16 hours.

[0237] In another aspect of the present invention, the temperature of the oxidation reaction is maintained at about 2°C to about 25°C, about 2°C to about 8°C, or about 20°C to about 25°C. In a 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.

[0238] In another 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.

[0239] In a further aspect, the concentration of the buffer is about 1 mM to about 500 mM, about 1 mM to about 300 mM, or about 50 mM to about 200 mM. In a preferred embodiment, the concentration of the buffer is about 100 mM.

[0240] In one aspect, the oxidation reaction is carried out at a pH of about 4.0 to about 8.0, about 5.0 to about 7.0, or about 5.5 to about 6.5. In a preferred embodiment, the pH is about 6.0.

[0241] In one embodiment, the activated GBS capsular polysaccharide is obtained by reacting about 0.5 mg / L to about 5.0 mg / mL of the isolated capsular polysaccharide with about 0.05 to about 0.3 molar equivalents of periodate at a temperature of about 20°C to 25°C.

[0242] In another embodiment, the activated GBS capsular polysaccharide is obtained by reacting about 0.5 mg / L to about 5.0 mg / mL of the isolated capsular polysaccharide with about 0.05 to about 0.3 molar equivalents of periodate at a temperature of about 2°C to 8°C.

[0243] 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 / diafiltration. For example, the activated capsular polysaccharide is purified by concentration and diafiltration using an ultrafiltration device.

[0244] In one embodiment, the degree of oxidation of the activated GBS capsular polysaccharide is 5 to 25, such as 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15. In a preferred embodiment, the degree of oxidation of the activated GBS capsular polysaccharide is 10 to 20, 11 to 19, 12 to 18, 13 to 17, or 14 to 16.

[0245] In another embodiment, the molecular weight of the activated GBS capsular polysaccharide is from about 5 kDa to about 1,000 kDa, such as from about 50 kDa to about 300 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 400 kDa, from about 300 kDa to about 700 kDa. In a preferred embodiment, the molecular weight of the activated GBS capsular polysaccharide is from about 75 kDa to about 400 kDa.

[0246] In one embodiment, the activated GBS capsular polysaccharide is optionally lyophilized 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. Then, the lyophilized activated capsular polysaccharide can be mixed with a solution containing a carrier protein.

[0247] In another embodiment, the activated GBS capsular polysaccharide is optionally mixed with a carrier protein in the presence of a sugar and lyophilized. 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. Then, the co-lyophilized polysaccharide and carrier protein can be resuspended in a solution and reacted with a reducing agent.

[0248] The activated GBS capsular polysaccharide can be conjugated to a carrier protein by a method comprising the following steps:

[0249] (a) Mixing the activated GBS capsular polysaccharide with the carrier protein,

[0250] (b) Reacting the mixed activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate.

[0251] For example, compared to reductive amination in an aqueous solution where the amount of unreacted (free) polysaccharide is significantly increased, conjugating the activated GBS capsular polysaccharide to a carrier protein by performing reductive amination in a polar aprotic solvent is suitable for maintaining a low level of free polysaccharide. In a preferred embodiment, steps (a) and (b) are performed in a polar aprotic solvent.

[0252] In one embodiment, step (a) includes dissolving freeze-dried GBS capsular polysaccharide in a solution containing a carrier protein and an aprotic polar solvent. In another embodiment, step (a) includes dissolving co-freeze-dried GBS capsular polysaccharide and a carrier protein in an aprotic polar solvent.

[0253] In one embodiment, the aprotic polar solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), sulfolane, dimethylformamide (DMF), and hexamethylphosphoramide (HMPA). In a preferred embodiment, the aprotic polar solvent is DMSO.

[0254] When steps (a) and (b) are carried out in an aqueous solution, steps (a) and (b) are carried out in a buffer in an aqueous medium, which is preferably selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine, or HEPB, with a pH of about 6.0 to about 8.5, about 7.0 to about 8.0, or about 7.0 to about 7.5. In a preferred embodiment, the buffer is PBS. In a preferred embodiment, the pH is about 7.3.

[0255] In one embodiment, the concentration of the activated GBS capsular polysaccharide in step (b) is about 0.1 mg / mL to about 10.0 mg / mL, about 0.5 mg / mL to about 5.0 mg / mL, or about 0.5 mg / mL to about 2.0 mg / mL. In a preferred embodiment, the concentration of the activated serotype GBS capsular polysaccharide in step (b) is about 0.1 mg / 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.

[0256] In a preferred embodiment, the initial ratio (weight to weight) of the activated serotype GBS capsular polysaccharide to the carrier protein is from 5:1 to 0.1:1, from 2:1 to 0.1:1, from 2:1 to 1:1, from 1.5:1 to 1:1, from 0.1:1 to 1:1, from 0.3:1 to 1:1, from 0.6:1 to 1:1. In a preferred embodiment, the initial ratio of the activated serotype GBS capsular polysaccharide to the 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.

[0257] In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride and zinc borohydride in the presence of a Bronsted acid or a Lewis acid, an amine borane such as pyridine borane, 2-methylpyridine borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i PrN-BH 3 , benzylamine-BH 3 , or 5-ethyl-2-methylpyridine borane (PEMB). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0258] In another embodiment, the amount of the reducing agent used in step (b) is from about 0.1 to about 10.0 molar equivalents, from about 0.5 to about 5.0 molar equivalents, or from about 1.0 to about 2.0 molar equivalents. In a preferred embodiment, the amount of the 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.

[0259] In a preferred embodiment, the duration of step (b) is from 1 hour to 60 hours, from 10 hours to 50 hours, from 40 hours to 50 hours, or from 42 hours to 46 hours. In a preferred embodiment, the duration of step (b) is about 44 hours.

[0260] In another embodiment, the reaction temperature of step (b) is maintained at 10°C to 40°C, 15°C to 30°C, or 20°C to 26°C. In a preferred embodiment, the reaction temperature in step (b) is maintained at about 23°C.

[0261] In a further embodiment, the method for manufacturing an immunogenic conjugate comprising a GBS capsular polysaccharide covalently linked to a carrier protein further comprises a step (step (c)) of capping (quenching) unreacted aldehydes by adding borohydride.

[0262] In one embodiment, the capping reagent is a borohydride selected from the group consisting of: sodium borohydride (NaBH 4 ), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, and magnesium borohydride. In a preferred embodiment, the capping reagent is sodium borohydride.

[0263] In another embodiment, the amount of borohydride used in step (c) is about 0.1 to about 10.0 molar equivalents, about 0.5 to about 5.0 molar equivalents, or about 1.0 to about 3.0 molar equivalents. In a preferred embodiment, the amount of borohydride used in step (c) is about 2.0 molar equivalents.

[0264] In a preferred embodiment, the borohydride used in step (c) is NaBH 4 , and its concentration is about 2.0 molar equivalents.

[0265] In one embodiment, the duration of step (c) is from 0.1 hour to 10 hours, from 0.5 hour to 5 hours, or from 2 hours to 4 hours. In a preferred embodiment, the duration of step (c) is about 3 hours.

[0266] In another embodiment, the reaction temperature in step (c) is maintained at about 15°C to about 45°C, about 15°C to about 30°C, or about 20°C to about 26°C. In a preferred embodiment, the reaction temperature of step (c) is maintained at about 23°C.

[0267] After the GBS capsular polysaccharide is conjugated to the carrier protein and capped, the polysaccharide-protein conjugate can be purified (enriched in terms of the amount of the polysaccharide-protein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration, precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration.

[0268] In another embodiment, the immunogenic conjugate contains 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 compared to the total amount of the GBS capsular polysaccharide. In a preferred embodiment, the immunogenic conjugate contains less than about 5% unreacted free sugar compared to the total amount of the GBS capsular polysaccharide.

[0269] In a preferred embodiment, the molecular weight of the GBS polysaccharide-protein conjugate is about 300 kDa to about 20,000 kDa, such as about 1,000 kDa to about 15,000 kDa, or about 1,000 kDa to about 10,000 kDa.

[0270] In another embodiment, the ratio (weight / weight) of the GBS capsular polysaccharide to the carrier protein in the conjugate is from about 0.5 to about 3.0. In one aspect, the ratio of the GBS capsular polysaccharide to the carrier protein in the conjugate is from about 0.5 to about 2.0, from about 0.5 to about 1.5, from about 0.5 to about 1.0, from about 1.0 to about 1.5, or from about 1.0 to about 2.0. In a preferred embodiment, the ratio of the GBS capsular polysaccharide to the carrier protein in the conjugate is from about 0.8 to about 1.0.

[0271] In another embodiment, the degree of conjugation of the conjugate is 2 to 15, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 15, 3 to 13, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 5 to 15, 5 to 10, 8 to 15, 8 to 12, 10 to 15, or 10 to 12. In a preferred embodiment, the degree of conjugation of the conjugate is 2 to 5.

[0272] In one aspect of the invention, the GBS capsular polysaccharide-protein conjugate is obtained by the reductive amination method described above. For example, in one aspect, the present disclosure provides a GBS capsular polysaccharide-protein conjugate comprising a polysaccharide conjugated to a carrier protein, which can be made or obtained by a method comprising the following steps:

[0273] (a) Reacting the isolated GBS capsular polysaccharide with an oxidizing agent;

[0274] (b) Quenching the oxidation reaction by adding a quenching agent to produce an activated GBS capsular polysaccharide;

[0275] (c) Mixing the activated GBS capsular polysaccharide with the carrier protein,

[0276] (d) Reacting the mixed activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate, and, optionally

[0277] (e) Capping unreacted aldehydes by adding sodium borohydride (NaBH 4 ).

[0278] In a preferred embodiment, steps (c) and (d) are carried out in DMSO.

[0279] In another aspect of the invention, the GBS capsular polysaccharide-protein conjugate of the invention is manufactured using reductive amination as described above, but 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) radical and N-chlorosuccinimide (NCS) are used as co-oxidants in the activation / oxidation step. See International Patent Application Publication No. WO 2014 / 097099, the entire content of which is incorporated herein by reference. In such an embodiment, the glycoconjugate from the GBS capsular polysaccharide is manufactured by oxidizing the primary alcohol of the sugar to an aldehyde using the TEMPO radical (using NCS as a co-oxidant) (hereinafter referred to as "TEMPO / NCS oxidation"), such as described in Example 7 and International Patent Application Publication No. WO 2014 / 097099. Thus, in one aspect, the conjugate of the GBS capsular polysaccharide is obtained by a method comprising the steps of: a) reacting the GBS capsular polysaccharide with TEMPO and NCS in a solvent to manufacture an activated sugar; and b) reacting the activated sugar with a carrier protein comprising one or more amino groups (hereinafter referred to as "TEMPO / NCS-reductive amination"). In one embodiment, the solvent may be an aqueous solvent or DMSO.

[0280] In one aspect, the GBS capsular polysaccharide-protein conjugate is obtained by the method described above. For example, in one aspect, the present disclosure provides a GBS capsular polysaccharide-protein conjugate comprising a polysaccharide conjugated to a carrier protein manufactured or obtained by a method comprising the steps of: a) reacting a sugar with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in a solvent to manufacture an activated sugar; and b) reacting the activated sugar with a carrier protein comprising one or more amino groups. In one embodiment, the solvent may be an aqueous solvent or DMSO.

[0281] Immunogenic composition

[0282] After the individual conjugates are purified, they can be combined to formulate the immunogenic composition of the invention, which can be used, for example, in a vaccine. The formulation of the immunogenic composition of the invention can be accomplished using methods recognized in the art.

[0283] An "immune response" to an immunogenic composition is the development by a subject of a humoral and / or cell-mediated immune response to a molecule (e.g., an antigen such as a protein or polysaccharide) present in the target composition. For the purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response and involves the production of antibodies that are specific for an antigen present in the immunogenic composition of the present invention, while a "cell-mediated immune response" is an immune response mediated by T-lymphocytes and / or other white blood cells. A "cell-mediated immune response" is induced by the presentation of antigenic epitopes associated 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 lymphocytes (CTLs). CTLs are specific for peptide or lipid antigens presented in association with proteins encoded by MHC or CD1 and expressed on the cell surface. CTLs assist in the induction and promotion of the intracellular destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves antigen-specific responses by helper T cells. Helper T cells act to assist in stimulating the functions of non-specific effector cells against cells displaying peptide antigens (associated with classical or non-classical MHC molecules) on their surface and to regulate their activities. A "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 immune response can be determined by a variety of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxicity assays, by assaying for T lymphocytes specific for an antigen in a sensitized subject, or by measuring cytokines produced by T cells in response to antigen restimulation. Such assays are well known in the art. See, for example, Erickson, A.L., et al., J. Immunol., 151(8):4189-4199 (1993); Doe, B., et al., Eur. J. Immunol. 24(10):2369-2376 (1994).

[0284] The term "immunogenic" refers to the ability of an antigen or vaccine to elicit an immune response (humoral or cell-mediated, or both).

[0285] As used herein, the terms "immunogenic amount", or "immunologically effective amount", or "dose" are used interchangeably and generally refer to the amount of an antigen or immunogenic composition (measured by standard assays known to those of skill in the art) sufficient to elicit an immune response (a cellular (T cell), or humoral (B cell or antibody), response, or both).

[0286] As used herein, "immune interference" or "substantial immune interference" means a statistically significant decrease in the immune response to an individual antigen in a multivalent or multicomponent vaccine as compared to the immune response to the same antigen administered as a monovalent vaccine.

[0287] A "protective" immune response refers to the ability of an immunogenic composition to elicit an immune response (humoral or cell-mediated) that protects a subject from infection. The protection provided need not be absolute, i.e., it is not required to completely prevent or eradicate the infection, if there is a statistically significant improvement as compared to a control group of subjects (e.g., infected animals that have not been vaccinated or administered the immunogenic composition). The protection may be limited to reducing the severity or rate of onset of the symptoms of the infection. Several assays are known in the art for determining whether an immune response is shown to be a "protective immune response". For example, an increase in antibody levels can be measured by a binding assay, such as the whole cell ELISA assay further described below. Other assays include measuring a functional antibody response, such as promoting the killing of bacteria, which can be tested with an opsonophagocytic assay (OPA) as described below. In certain instances, a "protective immune response" can include an increase in antibody levels specific for a particular antigen by two-fold or four-fold in at least 50% of the subjects. In another instance, a "protective immune response" can include a reduction in the number of bacteria by at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, or more.

[0288] The amount of a specific conjugate in a composition is generally calculated based on all of the polysaccharides (conjugated and unconjugated) used for the conjugate. For example, in a GBS capsular polysaccharide dose of 100 mcg / ml, a GBS capsular polysaccharide conjugate with 20% free polysaccharide will have approximately 80 mcg / ml of conjugated GBS capsular polysaccharide and approximately 20 mcg / ml of unconjugated GBS capsular polysaccharide. When calculating the dose of the conjugate, the contribution of the protein carrier to the conjugate is generally not considered. The amount of the conjugate can vary according to the streptococcal serotype. Generally, each dose will include from about 0.01 mg / ml to about 100 mcg / ml of each polysaccharide, particularly from about 1 mcg / ml to about 70 mcg / ml, and more particularly from about 5 mcg / ml to about 50 mcg / ml. The "immunogenic amount" of the different polysaccharide components in an immunogenic composition can be different and can each include 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 15 mcg / ml, about 20 mcg / ml, about 25 mcg / ml, about 30 mcg / ml, about 40 mcg / ml, about 50 mcg / ml, about 60 mcg / ml, about 70 mcg / ml, about 80 mcg / ml, about 90 mcg / ml, or about 100 mcg / ml of any particular polysaccharide antigen. Unless otherwise specified, the dose or immunogenic amount of a multivalent immunogenic composition will refer to the dose of each polysaccharide. For example, a dose of a hexavalent immunogenic composition of 10 mcg / ml will contain 10 mcg / ml of each of the six polysaccharides.

[0289] The potency of an antigen as an immunogen can be determined by measuring the level of B cell activity (by measuring the level of circulating antibodies specific for the antigen in serum using immunoassays, immunoprecipitation assays, functional antibody assays such as in vitro opsonophagocytosis assays, and many other assays known in the art). Another method for measuring the potency of an antigen as a T cell immunogen can be determined by proliferation assays, by cytolytic assays such as chromium release assays to measure the ability of T cells to lyse their specific target cells. In addition, in the present invention, an "immunogenic amount" can also be defined by measuring the serum level of antigen-specific antibodies induced after administration of the antigen, or by measuring the ability of the induced antibodies to enhance the opsonophagocytic capacity of specific white blood cells as described herein. The level of protection of the immune response can be measured by challenging the immunized host with the injected antigen. For example, if the antigen required for the immune response is a bacterium, the level of protection induced by the "immunogenic amount" of the antigen can be measured by detecting the percentage of survival or death of an animal challenged with bacterial cells. In one embodiment, the amount of protection can be determined by measuring at least one symptom associated with the bacterial infection (such as fever associated with the infection). The amount of each antigen in a multi-antigen or multi-component vaccine or immunogenic composition will vary relative to each other component and can be determined by methods known to those skilled in the art. Such methods can include, for example, procedures for measuring immunogenicity and / or in vivo potency.

[0290] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen (such as a microorganism or a component thereof) that 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 infection by administering the immunogenic composition via a systemic percutaneous or mucosal route. These administrations can include injection via intramuscular (i.m.), intraperitoneal (i.p.), intradermal (i.d.), or subcutaneous routes; administration via a patch or other percutaneous delivery device; or mucosal administration to the oral / digestive tract, respiratory tract, or urogenital tract. In one embodiment, the immunogenic composition can be used to manufacture a vaccine or to elicit polyclonal or monoclonal antibodies that can be used for passive protection or treatment of animals.

[0291] In one aspect, the present invention relates to an immunogenic composition comprising an effective amount of at least one polysaccharide, oligosaccharide, polysaccharide-protein conjugate, or a bioequivalent thereof as described herein. For example, in one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the capsular polysaccharide is selected from the group consisting of Streptococcus agalactiae serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX, and wherein the sialic acid level of the capsular polysaccharide is greater than about 60%. In another example, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae 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, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae 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 immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae 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 immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae 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 Streptococcus agalactiae 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 Streptococcus agalactiae serotypes Ia, Ib, II, III, and IV. In another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae 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, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae serotypes Ia, Ib, II, III, IV, and V.

[0292] In one embodiment, the immunogenic composition of the present invention comprises Streptococcus agalactiae of 2 to 10 different serotypes. Thus, in one embodiment, the immunogenic composition of the present 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 another embodiment, the immunogenic composition is a 7-valent GBS conjugate composition. In another embodiment, the immunogenic composition is an 8-valent GBS conjugate composition.

[0293] Although the prior art teaches the use of 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 experiences immune interference, especially with respect to the use of serotype V in a multivalent composition (see International Patent Application Publication No. WO 2012 / 035519), the present invention does not show any significant immune interference when using four or more GBS antigens and using serotype V in a multivalent composition. Thus, the present invention relates to a multivalent immunogenic composition comprising a polysaccharide-protein conjugate, the polysaccharide-protein conjugate 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, wherein the composition does not have significant immune interference. In a particular embodiment, the immunogenic composition comprises GBS capsular polysaccharide serotype V.

[0294] The polysaccharide-protein conjugate may comprise the same or different protein carriers. In one embodiment, the conjugate comprises the same protein carrier and the sugars are conjugated to the same molecule of the protein carrier (the carrier molecule has 2 or more different polysaccharides conjugated thereto) [see, for example, International Patent Application Publication No. WO 2004 / 083251]. In another embodiment, the polysaccharides are independently conjugated to different molecules of the protein carrier (each molecule of the protein carrier has only one type of polysaccharide conjugated thereto). In this embodiment, the capsular sugars are described as being conjugated individually to the carrier protein.

[0295] The optimal component amounts of a particular immunogenic composition can be determined by standard studies that involve observing the appropriate immune response of subjects. After the initial vaccination, the subjects may receive one or more appropriately spaced booster immunizations.

[0296] In addition to the multiple capsular polysaccharide-protein conjugates, the immunogenic compositions of the invention may further include one or more preservatives. The FDA requires that biologic products in multi-dose vials contain preservatives, with only a few exceptions. The invention contemplates the use of such multi-dose vials. Vaccine products containing preservatives include vaccines containing benzethonium chloride (anthrax), 2-phenoxyethanol (DTaP, HepA, Lyme, Polio (parenteral)), and phenol (Pneumo, typhoid (parenteral)). Preservatives approved for injectable drugs include, for example, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, phenol, and phenylmercuric nitrate.

[0297] In another aspect, the invention relates to a composition comprising at least one of any of the polysaccharides described herein and a pharmaceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, nonionic detergent, free radical oxidation inhibitor, diluent, or carrier, or a mixture thereof.

[0298] The immunogenic composition optionally may include one or more physiologically acceptable buffers selected from, but not limited to, HEPES, PIPES, MES, Tris (tromethamine), phosphates, acetates, borates, citrates, glycine, histidine, and succinates. In a preferred embodiment, the buffer is histidine.

[0299] 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, and most preferably about 20 mM.

[0300] In a preferred embodiment, the immunogenic composition comprises histidine at a concentration of about 20 mM.

[0301] In some embodiments, the formulation is buffered to a pH in the range of about 5.0 to about 7.1, such as 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 6.3 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, optimally about 6.5.

[0302] The immunogenic composition may optionally comprise one or more nonionic 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, BRIJ 58, BRIJ 35, and others such as TRITON X-100; TRITON X-114, NP40, SPAN 85, and the PLURONIC series of nonionic surfactants (e.g., PLURONIC 121). In one embodiment, the immunogenic composition comprises polysorbate-80 or polysorbate 40, preferably polysorbate-80 (PS80).

[0303] In one embodiment, the immunogenic composition comprises a surfactant at a concentration of from about 0.001% to about 2% (v / w), from about 0.001% to about 1%, from about 0.001% to about 0.5%, from about 0.001% to about 0.1%, from about 0.001% to about 0.05%, from about 0.001% to about 0.01%, from about 0.001% to 0.005%, from about 0.005% to about 2%, from about 0.005% to about 1%, from about 0.005% to about 0.5%, from about 0.005% to about 0.1%, from about 0.005% to about 0.05%, from about 0.005% to about 0.01%, from about 0.01% to about 2%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, from about 0.01% to about 0.1%, from about 0.01% to about 0.05%, from about 0.01% to about 0.04%, from about 0.01% to about 0.03%, from about 0.015% to about 2%, from about 0.015% to about 1%, from about 0.015% to about 0.5%, from about 0.015% to about 0.1%, from about 0.015% to about 0.05%, from about 0.015% to about 0.04%, from about 0.015% to about 0.03%, from about 0.02% to about 2%, from about 0.02% to about 1%, from about 0.02% to about 0.5%, from about 0.02% to about 0.1%, from about 0.02% to about 0.05%, from about 0.02% to about 0.04%, from about 0.02% to about 0.03%, from about 0.05% to about 2%, from about 0.05% to about 1%, from about 0.05% to about 0.5%, from about 0.05% to about 0.1%, from about 0.1% to about 2%, from about 0.1% to about 1%, from about 0.1% to about 0.5%, or from about 0.1% to about 0.25%. In a preferred embodiment, the immunogenic composition comprises a surfactant at a concentration of from about 0.01% to about 0.03%, and most preferably about 0.02%.

[0304] In another embodiment, the immunogenic composition comprises polysorbate-80 at a concentration of from about 0.001% to about 2% (preferably up to about 0.25%) or polysorbate 40 at a concentration of from about 0.001% to 1% (preferably up to about 0.5%).

[0305] In a preferred embodiment, the immunogenic composition comprises PS80 at a concentration of about 0.02%.

[0306] A pharmaceutically acceptable carrier is not to be confused with a "carrier protein" which is used to link the carbohydrate of the present invention to a protein and modify the immune response to the carbohydrate. To avoid confusion with the carrier protein described herein, the term pharmaceutically acceptable diluent will be preferred to pharmaceutically acceptable carrier, although these terms may be used interchangeably on occasion. The term "pharmaceutically acceptable carrier" means a carrier approved by a regulatory agency of the federal or state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals (including humans and non-human mammals). The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition 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 solutions, phosphate buffered saline, adjuvant suspensions, aqueous glucose and glycerol solutions, and combinations thereof can be used as liquid carriers, especially for injectable solutions. The pharmaceutically acceptable diluent can further include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers which enhance the shelf life or effectiveness in the body. The preparation and use of pharmaceutically acceptable diluents are known in the art. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. In one embodiment, the diluent is 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. In a preferred embodiment, the diluent is an aluminum-based adjuvant suspension such as an aluminum phosphate suspension.

[0307] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinitol, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. In a preferred embodiment, the excipient is NaCl.

[0308] In one embodiment, the immunogenic composition comprises an excipient at a concentration of from about 10 mM to about 500 mM, from about 10 mM to about 450 mM, from about 10 mM to about 400 mM, from about 10 mM to about 350 mM, from about 10 mM to about 300 mM, from about 10 mM to about 250 mM, from about 10 mM to about 200 mM, from about 10 mM to about 150 mM, from about 10 mM to about 100 mM, from about 10 mM to about 50 mM, from about 10 mM to about 30 mM, from about 10 mM to about 20 mM, from 20 mM to about 500 mM, from about 20 mM to about 450 mM, from about 20 mM to about 400 mM, from about 20 mM to about 350 mM, from about 20 mM to about 300 mM, from about 20 mM to about 250 mM, from about 20 mM to about 200 mM, from about 20 mM to about 150 mM, from about 20 mM to about 100 mM, from about 20 mM to about 50 mM, from about 20 mM to about 30 mM, from 50 mM to about 500 mM, from about 50 mM to about 450 mM, from about 50 mM to about 400 mM, from about 50 mM to about 350 mM, from about 50 mM to about 300 mM, from about 50 mM to about 250 mM, from about 50 mM to about 200 mM, from about 50 mM to about 150 mM, from about 50 mM to about 100 mM, from about 100 mM to about 500 mM, from about 100 mM to about 450 mM, from about 100 mM to about 400 mM, from about 100 mM to about 350 mM, from about 100 mM to about 300 mM, from about 100 mM to about 250 mM, from about 100 mM to about 200 mM, from about 100 mM to about 150 mM, from about 150 mM to about 500 mM, from about 150 mM to about 450 mM, from about 150 mM to about 400 mM, from about 150 mM to about 350 mM, from about 150 mM to about 300 mM, from about 150 mM to about 250 mM, from about 150 mM to about 200 mM, from about 200 mM to about 500 mM, from about 200 mM to about 450 mM, from about 200 mM to about 400 mM, from about 200 mM to about 350 mM, from about 200 mM to about 300 mM, from about 200 mM to about 250 mM, from about 250 mM to about 500 mM, from about 250 mM to about 450 mM, from about 250 mM to about 400 mM, from about 250 mM to about 350 mM, from about 250 mM to about 300 mM, from about 300 mM to about 500 mM, from about 300 mM to about 450 mM, from about 300 mM to about 400 mM, from about 300 mM to about 350 mM, from about 350 mM to about 500 mM, from about 350 mM to about 450 mM, from about 350 mM to about 400 mM, from about 400 mM to about 500 mM, from about 400 mM to about 450 mM, or from about 450 mM to about 500 mM. In a preferred embodiment, the immunogenic composition comprises an excipient at a concentration of from about 10 mM to about 250 mM, most preferably about 150 mM.

[0309] In a preferred embodiment, the excipient is NaCl at a concentration of about 150 mM.

[0310] If desired, the composition may also contain small amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents. These compositions can be in the form of solutions, suspensions, emulsions, lyophilized powders or cakes, etc. The formulation should be suitable for the mode of administration. Any conventional medium or reagent can be considered for use in the immunogenic compositions of the present invention, except those that are incompatible with the active ingredient.

[0311] 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, palatinitol, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim 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 contains additional excipients. In one such embodiment, the additional excipient is mannitol or glycine.

[0312] In another embodiment, the lyophilized composition contains from about 1% (w / v) to about 10% (w / v) of at least one sugar, 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 contains more than about 5.5% (w / v) of at least one excipient, such as more than about 7.0% (w / v). In another embodiment, the lyophilized composition contains from about 1% (w / v) to about 10% (w / v) of additional excipients, 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 contains from about 1% (w / v) to about 10% (w / v) of at least one excipient and from about 1% (w / v) to about 10% (w / v) of additional excipients.

[0313] In 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.

[0314] In one embodiment, the composition comprises the isolated polysaccharide and carrier molecule described herein. Suitable carrier molecules can include proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactivated virus particles. Examples of particulate carriers include those derived from polymethylmethacrylate polymers and those derived from poly(lactide-co-glycolide) (referred to as PLG).

[0315] The immunogenic compositions of the invention may further comprise one or more additional "immunomodulators", which are agents that disrupt or alter the immune system such that an up- or down-regulation of humoral and / or cell-mediated immunity can be observed. In certain embodiments, up-regulation of the humoral and / or cell-mediated branches of the immune system is preferred. Among them, some examples of immunomodulators include, for example, the adjuvants or cytokines 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 an antigen as further described herein.

[0316] 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.); Adjuvants; saponins; Quil A or other saponin moieties; monophosphoryl lipid A; and Avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions that can be used as adjuvants in the vaccines of the present invention include MF59 (U.S. Patent No. 6,299,884) (containing 5% squalene, 0.5% polysorbate-80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE), which is formulated into submicron particles using a microfluidizer such as Model 110Y Microfluidizer (Microfluidics, Newton, MA)), and SAF (containing 10% squalene, 0.4% polysorbate-80, 5% pluronic block polymer L121, and thr-MDP, which is microfluidized into a submicron emulsion or shaken to produce a larger particle size emulsion); modified SEAM62 (containing 5% (v / v) squalene (Sigma), 1% (v / v) detergent (ICI Surfactants), 0.7% (v / v) polysorbate 80 detergent (ICI Surfactants), 2.5% (v / v) ethanol, 200 μg / ml Quil A, 100 μg / ml cholesterol, and 0.5% (v / v) lecithin); and modified SEAM 1 / 2 (containing 5% (v / v) squalene, 1% (v / v) detergent, 0.7% (v / v) polysorbate 80 detergent, 2.5% (v / v) ethanol, 100 μg / ml Quil A, and 50 μg / ml cholesterol).

[0317] Suitable adjuvants for enhancing the immune response also include, but are not limited to, MPL described in U.S. Patent No. 4,912,094 TM (3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT). Also suitable as adjuvants are synthetic lipid A analogs, or aminoalkyl glucosamine phosphate compounds (AGP), or their derivatives or analogs, which are available from Corixa (Hamilton, MT) and are described in U.S. Patent No. 6,113,918. One such AGP is 2-[(R)-3-tetradecanoyloxy-tetradecanoyl-amino]ethyl 2-deoxy-4-O-phosphoryl-3-O-[(R)-3-tetradecanoyloxy-tetradecanoyl]-2-[(R)-3-tetradecanoyloxy-tetradecanoyl-amino]-β-D-glucopyranoside, which is also known as 529 (formerly known as RC529). This 529 adjuvant is formulated as an aqueous form (AF) or a stable emulsion (SE).

[0318] Other adjuvants include cyclodextrin derivatives (U.S. Patent No. 6,165,995); polyanionic polymers (U.S. Patent 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-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamine (MTP-PE); Amphigen; Avridine; L121 / squalene; D-lactide-polylactide / glycoside; pluronic polyols; killed Bordetella; saponins, such as Stimulon described in U.S. Patent No. 5,057,540 TM QS-21 (Antigenics, Framingham, MA.); Mycobacterium tuberculosis; bacterial lipopolysaccharides; synthetic polynucleotides, such as oligonucleotides containing CpG motifs (e.g., U.S. Patent No. 6,207,646); IC-31 described in European Patent Nos. 1,296,713 and 1,326,634 (Intercell AG, Vienna, Austria); pertussis toxin (PT) or mutants thereof, cholera toxin or mutants thereof (e.g., U.S. Patent Nos. 7,285,281, 7,332,174, 7,361,355 and 7,384,640); or Escherichia coli heat-labile toxin (LT) or mutants thereof, especially LT-K63, LT-R72 (e.g., U.S. Patent Nos. 6,149,919, 7,115,730 and 7,291,588).

[0319] Other "immunomodulators" that may be included in the vaccine include, for example, one or more of the following: interleukin 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, for example, U.S. Patent 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, for example, U.S. Patent No. 5,078,996 and ATCC accession number 39900); macrophage colony-stimulating factor (M-CSF); granulocyte colony-stimulating factor (G-CSF); or tumor necrosis factor α and β. Other adjuvants that may be used 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, for example, 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, ICAM (e.g., 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 angiogenic growth factors, nerve growth factors, fibroblast growth factors, epidermal growth factors, 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).

[0320] It should be understood that the decision whether to use an immunomodulator and / or an adjuvant, or the choice of which immunomodulator and / or adjuvant to use, will depend on the subject to whom the vaccine or immunogenic composition is to be administered, the route of injection, and the number of injections to be given. For example, if the subject has been naturally exposed to the pathogen, an adjuvant may not be needed because the vaccine antigen may effectively induce a memory response. In some embodiments, the immunogenic composition will include one or more adjuvants. In one embodiment, the immunogenic composition comprises an aluminum-based adjuvant. In one such embodiment, the aluminum adjuvant is aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphate. In a particular embodiment, the adjuvant is aluminum phosphate. In another embodiment of the invention, the immunogenic composition comprises QS-21 as an adjuvant.

[0321] In one embodiment, the immunogenic composition comprises an adjuvant at a concentration of from about 0.1 mg / ml to about 1.0 mg / ml, from 0.1 mg / ml to about 0.9 mg / ml, from 0.1 mg / ml to about 0.8 mg / ml, from 0.1 mg / ml to about 0.7 mg / ml, from 0.1 mg / ml to about 0.6 mg / ml, from 0.1 mg / ml to about 0.5 mg / ml, from 0.1 mg / ml to about 0.4 mg / ml, from 0.1 mg / ml to about 0.3 mg / ml, from 0.1 mg / ml to about 0.2 mg / ml, from 0.25 mg / ml to about 0.95 mg / ml, from 0.25 mg / ml to about 0.85 mg / ml, from 0.25 mg / ml to about 0.75 mg / ml, from 0.25 mg / ml to about 0.65 mg / ml, from 0.25 mg / ml to about 0.55 mg / ml, from 0.25 mg / ml to about 0.45 mg / ml, from 0.25 mg / ml to about 0.35 mg / ml, from 0.5 mg / ml to about 1.0 mg / ml, from 0.5 mg / ml to about 0.9 mg / ml, from 0.5 mg / ml to about 0.8 mg / ml, from 0.5 mg / ml to about 0.75 mg / ml, from 0.5 mg / ml to about 0.7 mg / ml, from 0.5 mg / ml to about 0.65 mg / ml, from 0.5 mg / ml to about 0.6 mg / ml, from 0.75 mg / ml to about 1.0 mg / ml, from 0.75 mg / ml to about 0.95 mg / ml, from 0.75 mg / ml to about 0.9 mg / ml and from 0.75 mg / ml to about 0.85 mg / ml. In a preferred embodiment, the immunogenic composition comprises an adjuvant at a concentration of from about 0.25 mg / ml to about 0.75 mg / ml, most preferably about 0.5 mg / ml.

[0322] In a preferred embodiment, the adjuvant is an aluminum-based adjuvant at a concentration of about 0.5 mg / ml. In such an embodiment, the aluminum-based adjuvant is aluminum phosphate or aluminum hydroxyphosphate.

[0323] In one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, buffer, surfactant, excipient and an optional adjuvant as described herein, wherein the composition is buffered to a pH of from about 6.0 to about 7.0.

[0324] In such an embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, buffer, surfactant, excipient and an optional adjuvant, wherein the composition is buffered to a pH of from about 6.0 to about 7.0, and wherein the sialic acid level of the capsular polysaccharide is greater than about 60%.

[0325] In certain embodiments, 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 from about 6.0 to about 7.0, and wherein the sialic acid level of the capsular polysaccharide is greater than about 60%.

[0326] In a preferred embodiment, the immunogenic composition comprises from about 5 mcg / ml to about 50 mcg / ml of a GBS polysaccharide-protein conjugate, from about 10 mM to about 25 mM of histidine, from about 0.01% to about 0.03% (v / w) of polysorbate-80, from about 10 mM to about 250 mM of sodium chloride, and optionally from about 0.25 mg / ml to about 0.75 mg / ml of aluminum as aluminum phosphate, wherein the sialic acid level of the capsular polysaccharide is greater than about 60%.

[0327] In such an 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 from about 6.0 to about 7.0, and wherein the conjugate comprises capsular polysaccharides from serotype IV of group B streptococcus (GBS) and at least one additional serotype selected from the group consisting of: Ia, Ib, II, III, V, VI, VII, VIII, and IX.

[0328] In certain embodiments, 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 from about 6.0 to about 7.0, and wherein the conjugate comprises capsular polysaccharides from serotype IV of group B streptococcus (GBS) and at least one additional serotype selected from the group consisting of: Ia, Ib, II, III, V, VI, VII, VIII, and IX.

[0329] In a preferred embodiment, the immunogenic composition comprises each from about 5 mcg / ml to about 50 mcg / ml of at least two GBS polysaccharide-protein conjugates, from about 10 mM to about 25 mM of histidine, from about 0.01% to about 0.03% (v / w) of polysorbate-80, from about 10 mM to about 250 mM of sodium chloride, and optionally from about 0.25 mg / ml to about 0.75 mg / ml of aluminum as aluminum phosphate, wherein the conjugate comprises capsular polysaccharides from serotype IV of group B streptococcus (GBS) and at least one additional serotype selected from the group consisting of: Ia, Ib, II, III, V, VI, VII, VIII, and IX.

[0330] Evaluation of the immunogenic composition

[0331] A variety of in vitro assays were used to evaluate the immunogenicity of the immunogenic compositions of the present invention. For example, in vitro opsonophagocytosis assays were performed by incubating a mixture of Streptococcus cells, heat-inactivated serum containing specific antibodies against the antigen under study, and an exogenous complement source. Opsonophagocytosis was performed during incubation of freshly isolated polymorphonuclear cells (PMNs) or differentiated effector cells such as HL60 with the antibody / complement / Streptococcus cell mixture. During opsonophagocytosis, bacterium cells coated with antibody and complement are killed. Colony forming units (cfu) of surviving bacteria recovered from opsonophagocytosis were determined by plating the assay mixture. The reported titer is the reciprocal of the highest dilution that results in 50% killing of the bacteria, as determined by comparison with an assay control.

[0332] Whole cell ELISA assays can also be used to evaluate the in vitro immunogenicity and surface exposure of antigens, where the target strain (Streptococcus agalactiae) is coated on a plate (e.g., a 96-well plate) and test sera from immunized animals are reacted with the bacterial cells. If antibodies specific for the test antigen react with surface-exposed epitopes of the antigen, this can be detected by standard methods known to those skilled in the art. Alternatively, flow cytometry can be used to measure the surface exposure of capsular polysaccharide antigens and the specificity of antibodies (including monoclonal antibodies).

[0333] Then, antigens showing the desired in vitro activity can be tested in an in vivo animal challenge model. In some embodiments, animals (e.g., mice) are immunized with the immunogenic compositions by immunization methods and routes known to those skilled in the art (e.g., intranasal, parenteral, oral, rectal, vaginal, transdermal, intraperitoneal, intravenous, subcutaneous, etc.). After immunizing the animals with the GBS immunogenic composition, the animals are challenged with a Streptococcus agalactiae strain and their resistance to streptococcal infection is analyzed.

[0334] In one embodiment, pathogen-free mice are immunized and challenged with Streptococcus agalactiae. For example, mice are immunized with one or more doses of the target antigen in the immunogenic composition. Subsequently, the mice are challenged with Streptococcus agalactiae and survival is monitored over time after the challenge.

[0335] Method of Use

[0336] As used herein, "immunocompromised" refers to a subject who suffers from a defect in the cellular and / or humoral arm of the immune system. Thus, degrees of immunodeficiency are contemplated that range from a slight impairment in the immune process to complete immunosuppression.

[0337] 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. Non-limiting examples of livestock include: alpacas, bison, camels, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

[0338] As used herein, "treatment" (including variations thereof such as "treat" or "treating") refers to any one or more of the following: (i) preventing infection or reinfection, as in traditional vaccines, (ii) reducing the severity of symptoms or eliminating symptoms, and (iii) substantially or completely eliminating the pathogen or condition of interest. Thus, treatment can act prophylactically (before infection) or therapeutically (after infection). In the present invention, prophylactic or therapeutic treatment can be used. According to a particular embodiment of the present invention, compositions and methods for treating (including prophylactically and / or therapeutically immunizing) a host animal against a microbial infection (e.g., a bacterium such as Streptococcus agalactiae) are provided. The methods of the present invention can be used to confer prophylactic and / or therapeutic immunity to a subject. The methods of the present invention can also be performed in a subject for biomedical research applications.

[0339] On the other hand, the present 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 present invention relates to a method of preventing or alleviating a disease or condition associated with Streptococcus agalactiae in a subject by administering to the subject an effective amount of the immunogenic composition described herein. In one aspect, the present invention relates to the use of the immunogenic composition described herein as a medicament. In one aspect, the present invention relates to the use of the immunogenic composition described herein in a method of inducing an immune response against GBS in a subject. In a particular embodiment, the subject is a female planning to become pregnant or a pregnant female. In one such embodiment, the pregnant female is in the third trimester of her pregnancy, such as at least 20 weeks of gestation or at least 27 weeks of gestation. In a preferred embodiment, the pregnant female is between 27 and 36 weeks of gestation. In another embodiment, the subject is an elderly adult such as an adult 50 years of age or older, 65 years of age or older, and 85 years of age or older. In another embodiment, the subject is immunocompromised. In one aspect, the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease. In a preferred embodiment, the Streptococcus agalactiae is S. agalactiae.

[0340] In one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate that comprises 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 a further 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 a particular embodiment, the conjugate comprises GBS serotypes Ia, Ib, II, III, and IV. In another 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 another 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 invention relates to an immunogenic composition that does not have immune interference.

[0341] The immunogenicity or effective amount of the immunogenic composition can be determined by conducting a dose-response study in which subjects are immunized with progressively increasing amounts of the immunogenic composition and the immune response is analyzed to determine the optimal dose. The starting point of the study can be inferred from immunization data in animal models. The dose can be varied according to the particular circumstances of the subject. The amount can be determined in routine tests by methods known to those skilled in the art.

[0342] An immunologically effective amount of the immunogenic composition is administered to the subject in a suitable number of doses to elicit an immune response. The dose can vary according to the particular condition of the subject such as age and weight. This amount can be determined in routine tests by methods known to those skilled in the art.

[0343] In one embodiment, patients administered with the immunogenic composition of the present invention show a reduced carriage rate of Streptococcus agalactiae. From the perspective of medical need, such a reduction in the carriage rate or an extended time interval as a non-carrier after administration of the immunogenic composition is significant. For example, after administration of a dose of the immunogenic composition of the present invention, a reduction in the overall Streptococcus agalactiae carriage rate of carriers can be evaluated. For example, one day before administration of the immunogenic composition, a group of adults aged 18 to 50 years can be screened for carriage by nasal, throat, axillary, rectal, perineal, and vaginal swabs, followed by culturing to determine their carriage state. Then, the immunogenic composition of the present invention is administered to this group, while another group receives a control. Nasal, throat, axillary, rectal, perineal, and vaginal swabs are continuously taken weekly for 12 weeks after administration of the immunogenic composition and monthly for up to 6 months, and compared with the placebo group. A primary endpoint is to compare the carriage rate of patients after administration of the immunogenic composition and after administration of the placebo in a 3-month period after immunization.

[0344] antibody

[0345] "Antibody" refers to an immunoglobulin molecule capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Unless otherwise specified in the context, this term as used herein is intended to include not only intact polyclonal or monoclonal antibodies, but also genetically engineered antibodies (e.g., chimeric, humanized, and / or derivatized to alter effector function, stability, and other biological activities) and fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain (ScFv) and domain antibodies, including shark and camel antibodies), and fusion proteins containing antibody moieties, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, provided that they exhibit the desired biological activity), and any other modified configurations of immunoglobulin molecules described herein, as well as antibody fragments including antigen recognition sites. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or their subclasses), and the antibody need not be of any particular class. Immunoglobulins can be classified into different classes according to the amino acid sequence of the constant domain of the antibody heavy chain. There are five major classes of immunoglobulins in the human body: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0346] "Antibody fragment" includes only a portion of a full antibody, where the portion preferably retains at least one, preferably most or all of the functions normally associated with that portion as present in the full antibody.

[0347] As used herein, "functional activity" or "functional antibody" of an antibody means that the antibody can specifically bind an antigen at least. Other functions are known in the art and may include other components of the immune system that can clear or kill pathogens (such as through opsonization, ADCC, or complement-mediated cytotoxicity). After antigen binding, any subsequent antibody functions can be mediated by the Fc region of the antibody. Antibody opsonophagocytosis assay (OPA) is an in vitro assay designed to measure in vitro Ig complement-assisted bacterial killing by effector cells (leukocytes), thus mimicking biological processes. Antibody binding can also directly inhibit the biological function of the antigen it binds. In some embodiments, "functional antibody" means an antibody having a function measured by killing bacteria in an animal efficacy model or an opsonophagocytic killing assay demonstrating that the antibody kills bacteria.

[0348] In one aspect, the present invention relates to an isolated antibody or fragment thereof that specifically binds to the polysaccharides described herein. As used herein, an "isolated" antibody means an antibody that has been identified and separated and / or recovered from the components of its natural environment. The contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In an exemplary embodiment, the antibody will be purified to (1) more than 95% by weight of the antibody (determined by the Lowry method), most preferably more than 99% by weight, (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotor cup sequencer, or (3) be homogeneous as analyzed by SDS-PAGE when stained with Coomassie blue or preferably silver stain under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells because at least one component of the natural environment of the antibody will not be present. However, isolated antibodies will generally be produced by at least one purification step.

[0349] An antibody that "specifically binds" or "is specific for" a particular polysaccharide or an epitope on a particular polysaccharide is an antibody that binds to that particular polysaccharide or an epitope on that particular polysaccharide and does not substantially bind to any other polysaccharide or polysaccharide epitope.

[0350] As used herein, "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. The label can itself be detectable (such as a radioisotope label or a fluorescent label), or, in the case of an enzyme label, can catalyze a chemical change in a detectable substrate compound or composition.

[0351] 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 present invention. In some embodiments, the antibodies are generated upon administration of the immunogenic compositions of the present invention to a subject. In some embodiments, the present invention provides purified or isolated antibodies against one or more antigens of the immunogenic compositions of the present invention. In some embodiments, the antibodies of the present invention are measured as being functional by killing bacteria in an animal efficacy model or via an 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, and cells or cell lines for producing the antibodies or antibody compositions of the present invention (such as hybridoma cells or other genetically engineered cell lines for recombinantly producing antibodies) and transgenic animals (using techniques well known to those skilled in the art).

[0352] The antibodies or antibody compositions of the present invention can be used to treat or prevent streptococcal infections, diseases or conditions associated with Streptococcus agalactiae in a subject, the method comprising generating a polyclonal or monoclonal antibody preparation, and using the antibody or antibody composition to confer passive immunity to the subject. The antibodies of the present invention can also be used in diagnostic methods, for example, to detect the presence or quantify one or more antigens of the immunogenic compositions of the present invention.

[0353] Antibody responses to repetitive structures (such as the polysaccharides of the present invention) can exhibit some unique characteristics. 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. Second, the repeating structure of longer polysaccharides can induce T cell-independent antibody responses. Thus, when a polysaccharide conjugated to a protein carrier having a T helper cell epitope is used, both T cell-independent and T cell-dependent antibody responses can be stimulated. Therefore, the immune response can be modified by appropriate selection of the polysaccharide size and whether a carrier protein is used.

[0354] Polyclonal antibodies

[0355] In certain embodiments, the anti-polysaccharide antibodies are polyclonal antibodies. As defined herein, polyclonal antibodies refer to a mixture of antibodies having different specificities, derived from a serum preparation and originating from different B cell clones. Methods for preparing and characterizing polyclonal antibodies are known in the art.

[0356] Polyclonal antibodies are raised in a subject (e.g., a mammal) by injection of one or more of the immunogens or immunogenic compositions described herein, and if desired, an adjuvant, buffer, and / or diluent. There are many animal species that can be used to produce specific antisera. Generally, the animals used to produce antiglycan polyclonal antisera are non-human primates, goats, sheep, rabbits, mice, rats, hamsters, or guinea pigs. Generally, the immunogen or immunogenic composition, with or without an adjuvant, is injected into the mammal by multiple injections. The immunogenic substance can include polysaccharides, oligosaccharides, the polysaccharides described herein, polysaccharide-protein conjugates, or larger immunogenic assemblies. Generally, starting 2 to 6 weeks after the first immunization, blood is collected from the immunized animal, allowed to clot, and the serum is harvested. This serum contains antiglycan polyclonal antibodies from the immunized animal and is commonly referred to as antiserum.

[0357] Monoclonal antibodies

[0358] Antiglycan monoclonal antibodies can be produced using known hybridoma techniques. Generally, producing monoclonal antibodies involves first immunizing a suitable target animal host with a selected immunogen (including polysaccharides, oligosaccharides, the polysaccharides of the present invention, or polysaccharide-protein conjugates). If desired, an adjuvant, buffer, and / or diluent can be included. The immunization is carried out in a manner sufficient to induce B lymphocytes to produce or express antibodies that specifically bind to the polysaccharide or its conjugate. Alternatively, the lymphocytes are immunized in vitro.

[0359] Then, the lymphocytes are fused with an immortalized cell line using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells. The source of the lymphocytes determines whether the monoclonal antibody is of human or animal origin. Generally, peripheral blood lymphocytes ("PBL") are used when human-derived antibodies and cells are needed, while spleen cells or lymph node cells are used when non-human mammalian-derived cells are needed.

[0360] The immortalized cell line is usually a transformed mammalian cell, especially myeloma cells of rodent, bovine, and human origin. Generally, rat or mouse myeloma cell lines are used. The hybridoma cells are cultured in a suitable medium, preferably one containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the medium for the hybridoma will usually include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which will prevent the growth of HGPRT-deficient cells.

[0361] The immortalized cell line is selected based on practical considerations such as the species of origin, fusion and growth characteristics. For example, suitable immortalized cell lines are those that fuse efficiently, support stable and high-level expression of the antibody by the selected antibody-producing cells, and are sensitive to media such as HAT medium. Examples of immortalized cell lines include murine myeloma lines. Human myelomas and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

[0362] Monoclonal antibodies are secreted by hybridoma cells into the culture medium. Then, the culture medium is analyzed for the presence of monoclonal antibodies that can recognize and bind to the polysaccharide. The anti-polysaccharide binding specificity of a particular monoclonal antibody produced by the hybridoma cells is determined by one of the many procedures well known in the art. For example, antibody binding specificity can be determined by immunoprecipitation, radioimmunoassay (RIA), Western blotting, enzyme-linked immunosorbent assay (ELISA), or surface plasmon resonance (e.g., Biacore). The precise epitope recognized by the monoclonal antibody is determined by epitope mapping. Such techniques and assays are well known in the art.

[0363] After identifying the antibody-producing hybridoma cells with the desired specificity, the clone is subcloned by limiting dilution and cultured using standard methods. Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells are grown in vivo in a mammal as ascites fluid. The monoclonal antibodies secreted by the subclone are isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0364] Alternatively, antibodies with the desired specificity and from the desired source species can be obtained by using a phage display library. In addition, examples of methods and reagents particularly suitable for generating and screening antibody display libraries can be found in the art.

[0365] Antibody Uses

[0366] In one aspect, the invention relates to the use of the immunogenic compositions described herein for the manufacture of GBS antibodies and / or antibody fragments. The polysaccharide-protein conjugates described herein and / or the antibodies generated therefrom can be used in a variety of immunoassay techniques known to those skilled in the art, including ELISA and microarray-related techniques. In addition, these reagents can be used to assess antibody responses, including, for example, serum antibody levels against the immunogenic polysaccharide conjugates. The assay methodologies of the invention can involve the use of labels such as fluorescence, chemiluminescence, radioactivity, enzymatic labels, or dye molecules, and / or a second immunological reagent for the direct or indirect detection of complexes between an antigen or antibody in a biological sample and the corresponding antibody or antigen bound to a solid support.

[0367] The antibodies or antibody fragments generated can also be used in passive immunotherapy or prophylactic therapy against streptococcal infections.

[0368] Method for producing polysaccharide

[0369] On the other hand, the present invention relates to a method for manufacturing the polysaccharides described herein. The method includes culturing GBS and collecting the polysaccharides produced by the bacterium. In one embodiment, the GBS includes Streptococcus agalactiae. The bacterium can be any strain of Streptococcus agalactiae. In a preferred embodiment, the bacterium is an encapsulated strain of Streptococcus agalactiae. Streptococcus agalactiae strains for use in the present invention include 090, A909 (ATCC accession number BAA-1138), 515 (ATCC accession number BAA-1177), B523, CJB524, MB4052 (ATCC accession number 31574), H36B (ATCC accession number 12401), S40, S42, MB 4053 (ATCC accession number 31575), M709, 133, 7357, PFEGBST0267, MB 4055 (ATCC accession number 31576), 18RS21 (ATCC accession number BAA-1175), S16, S20, V8 (ATCC accession number 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB 4082 (ATCC accession number 31577), M132, 110, M781 (ATCC accession number BAA-22), D136C(3) (ATCC accession number 12403), M782, S23, 120, MB 4316 (M-732; ATCC accession number 31475), M132, K79, COH1 (ATCC accession number BAA-1176), PFEGBST0563, 3139 (ATCC accession number 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession number BAA-23), CJB112, 2603V / R (ATCC accession number BAA-611), NCTC 10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B4589, B 4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62 and IT-PW-64.

[0370] The polysaccharides described herein can be produced by culturing GBS in a suitable medium. Suitable media can include Columbia broth. The medium can contain dextrose, hemin, and / or glucose. Preferably, the medium contains Columbia broth and dextrose. When Streptococcus agalactiae is cultured using Columbia broth and dextrose, the culture temperature is preferably from 20 to 40 °C, more preferably 37 °C. In a preferred embodiment, the bacteria are cultured under aerobic conditions. In another preferred embodiment, the bacteria are cultured for 12 to 60 hours.

[0371] The polysaccharides can be collected from the resulting culture using methods known in the art for collecting target substances from cultures (such as, for example, heating, enzymatic treatment, centrifugation, precipitation, treatment with activated carbon, and / or filtration). (See, for example, U.S. Patent Application Publication Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498; International Patent Application Publication No. WO 2008 / 118752). In one embodiment, the culture containing the bacteria and polysaccharides is centrifuged and treated enzymatically, such as, for example, with lysozyme, ribonuclease, deoxyribonuclease, pronase, mutanolysin, and combinations 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. Then, a suitable organic solvent is further added to the supernatant to precipitate the polysaccharides, and the polysaccharides can be collected by centrifugation. More specifically, the polysaccharides described herein can be obtained by the following steps: ethanol is added to the supernatant from which the bacteria have been removed to a final concentration of about 25% by volume, the precipitate containing proteins is removed by centrifugation, ethanol is further added thereto to a final concentration of about 75% by volume, and then the precipitate is collected by centrifugation. The resulting precipitate can be dried with nitrogen. The resulting precipitate is resuspended in Tris and 0.05% sodium azide.

[0372] Another aspect of the present invention provides a novel method for separating most of the intact high molecular weight CP and retaining N- and O-acetyl groups using organic reagents such as derivatized hydroxylamine compounds. Since this method does not lyse cells, the CP separated by centrifugation is minimally contaminated with intracellular components and can result in a higher total yield. In addition, since the Group B antigen impurities have multiple phosphodiester bonds, these reagents cleave the Group B antigen impurities into very small fragments that can be easily removed by filtration.

[0373] In one embodiment, the CP is isolated by reacting hydroxylamine with a cell paste of bacteria containing capsular polysaccharide. In certain embodiments, the method further includes a centrifugation step. In another embodiment, the method further includes a filtration step. In another embodiment, the bacteria containing capsular polysaccharide are 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.

[0374] In one aspect of the invention, the hydroxylamine can be those listed in Table 2 of Example 2. In a preferred embodiment, 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.

[0375] In one aspect of the invention, the concentration of the hydroxylamine is from about 5 mM to about 200 mM, such as 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, from 10 mM to about 200 mM, such as 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, from about 25 mM to about 200 mM, from about 25 mM to about 150 mM, from about 25 mM to about 100 mM, from about 25 mM to about 75 mM, from about 25 mM to about 50 mM, from about 50 mM to about 200 mM, from about 50 mM to about 150 mM, from 50 mM to about 100 mM, and from about 50 mM to about 75 mM.

[0376] In another aspect, the pH of the reaction is maintained at about 5.5 to about 9.5, such as 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.

[0377] In another aspect of the present invention, the reaction occurs at a temperature of from about 20°C to about 85°C, such as from about 20°C to about 80°C, from about 20°C to about 75°C, from about 20°C to about 70°C, from about 20°C to about 65°C, from about 20°C to about 60°C, from about 20°C to about 55°C, from about 20°C to about 50°C, from about 25°C to about 85°C, from about 25°C to about 80°C, from about 25°C to about 75°C, from about 25°C to about 70°C, from about 25°C to about 65°C, from about 25°C to about 60°C, from about 25°C to about 55°C, from about 25°C to about 50°C, from about 30°C to about 85°C, from about 30°C to about 80°C, from about 30°C to about 75°C, from about 30°C to about 70°C, from about 30°C to about 65°C, from about 30°C to about 60°C, from about 30°C to about 55°C, from about 30°C to about 50°C, from about 35°C to about 85°C, from about 35°C to about 80°C, from about 35°C to about 75°C, from about 35°C to about 70°C, from about 35°C to about 65°C, from about 35°C to about 60°C, from about 35°C to about 55°C, from about 40°C to about 85°C, from about 40°C to about 80°C, from about 40°C to about 75°C, from about 40°C to about 70°C, from about 40°C to about 65°C, from about 40°C to about 60°C, from about 45°C to about 85°C, from about 45°C to 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.

[0378] In another aspect, the reaction time is from about 10 hours to about 90 hours, such as 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 75 hours, from about 15 hours to about 70 hours, from about 15 hours to about 60 hours, from about 15 hours to about 50 hours, from about 15 hours to about 40 hours, from about 15 hours to about 30 hours, from 15 hours to about 20 hours, such as from about 20 hours to about 90 hours, from about 20 hours to about 85 hours, from about 20 hours to about 80 hours, from about 20 hours to about 75 hours, from about 20 hours to about 70 hours, from about 20 hours to about 60 hours, from about 20 hours to about 50 hours, from about 20 hours to about 40 hours, from about 20 hours to about 30 hours, and from about 20 hours to about 25 hours.

[0379] Alternatively, in another embodiment of the present invention, the polysaccharide is chemically synthesized. The polysaccharide can be synthesized by chemical methods according to conventional methods.

[0380] In another embodiment of the present invention, the polysaccharide is produced by expression after cloning and expressing the biosynthetic pathway for producing the polysaccharide in an alternative host. For example, a host cell can be modified to produce a polysaccharide having a structure similar to the polysaccharides described herein, wherein the repeating unit of the polysaccharide produced in the host cell is partially identical to the repeating unit of the polysaccharides described herein. For example, compared to the repeating unit of the polysaccharides described herein, if the repeating unit of the polysaccharide has a missing branch, is heterogeneous in size, and / or is heterogeneous in the branching arrangement, the polysaccharide is structurally similar to the polysaccharides described herein. Preferably, the host cell is a bacterial host cell. Examples

[0381] The following examples illustrate some embodiments of the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to fully define the conditions and scope of the present invention. It should be understood that when typical reaction conditions (e.g., temperature, reaction time, etc.) are given, conditions above and below the specified range can also be used, although it is usually less convenient. Unless otherwise indicated, all parts and percentages mentioned herein are by weight and all temperatures are in degrees Celsius.

[0382] Furthermore, unless otherwise specified in detail, the following examples are carried out using standard techniques well known and conventional to those skilled in the art. As described above, the following examples are for illustrative purposes and should not limit the scope of the present invention in any way.

[0383] Example 1: Preparation of a polysaccharide-protein conjugate with de-O-acetylated polysaccharide

[0384] Streptococcus agalactiae strains of each serotype were fermented in submerged culture in a pH-controlled defined medium. The procedures and media used were optimized experimentally and are an extension of the basic techniques described previously by von Hunolstein, C. et al., Appl. Micro. Biotech. 38(4):458-462 (1993). The capsular polysaccharide was removed from the cells by treatment with NaOH. After clarification, a series of UF / DF, precipitation, and carbon filtration steps were carried out to produce the purified polysaccharide. See, for example, U.S. Patent No. 8,652,480. Reductive amination chemistry was used to conjugate the activated polysaccharide with CRM 197Conjugation. See, for example, U.S. Patent No. 5,360,897.

[0385] Example 2. Isolation of O-acetylated polysaccharide

[0386] The cell paste from GBS capsular polysaccharide (CP) serotype Ia obtained after heat killing and centrifugation of the 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 an aqueous hydroxyl amine O-sulfonic acid solution to a final concentration of 10 mM. The pH of the suspension was measured to be approximately 5.8. The suspension was stirred at 55 °C for 72 hours. Thereafter, the suspension was centrifuged at approximately 10,000 rpm and the supernatant was collected. The molecular weight and yield of the supernatant containing the crude cleaved CP were analyzed. The remainder was purified by diafiltration using water for injection (WFI) through a 30 kDa MWCO membrane. The molecular weight of the purified polysaccharide was further analyzed by size exclusion chromatography combined with a multiangle light scattering detector (SEC-MALS) (Table 1).

[0387] Table 1. Purification of GBS serotype Ia by diafiltration

[0388]

[0389] The activities of several hydroxylamines (both nitrogen- and oxygen-substituted compounds) were screened using the above method. The yield was calculated by gel permeation chromatography (GPC-MALS) of the crude supernatant combined with multi-angle light scattering detection (MALS), using a refractive index (RI) response and a square of the specific refractive index increment (dn / dc) value of 0.135. The yield depends on the optimization of the type of hydroxylamine and conditions such as concentration, temperature, and reaction time (see Table 2). Generally, increased hydroxylamine concentration, higher temperature, and longer reaction time result in higher yields.

[0390] Table 2. Screening of various hydroxylamines and optimization of conditions for GBS capsular polysaccharide serotype Ia

[0391]

[0392]

[0393]

[0394]

[0395] It was found that substituted and unsubstituted hydroxylamines are very effective in releasing GBS capsular polysaccharide from the cell wall. This method allows the isolation of high molecular weight CPS and preserves 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 to 8; temperature - 50 °C; time - 24 hours).

[0396] Table 3. GBS CP Release Data Using Dibenzylhydroxylamine

[0397] GBS type Ia Ib III Polysaccharide release yield 86％ 81％ 46％ Total purification yield 63％ 54％ 30％ Molecular weight (Mw) 330 kDa 212 kDa 171 kDa O-acetylation (NMR) NA 31％ 37％ N-acetyl (NMR) 106％ 104％ 87％

[0398] NA – Serotype Ia is not O-acetylated

[0399] Due to the poor solubility of dibenzylhydroxylamine in water, alternative derivatives of hydroxylamine that are water-soluble and have similar or higher activity to dibenzylhydroxylamine are desired. After screening several compounds, diethylhydroxylamine was found to be a good choice. The data are shown in Table 4 ([diethylhydroxylamine] - 100 mM; pH - 7 to 8; temperature - 60 °C; time - 19 hours).

[0400] Table 4. GBS CP Release Data Using Diethylhydroxylamine

[0401]

[0402]

[0403] Hydroxylamine (NH 2 -OH) was also found to be effective in lysing CPS from the cell wall. The data are shown in Table 5 ([hydroxylamine] - 100 mM; pH - 7 to 7.5; temperature - 65 °C; time - 17 hours). In the case of serotype III, the yield after 17 hours was 54%; however, the yield increased to 70% after three and a half days.

[0404] Table 5. GBS CP Release Data Using Hydroxylamine

[0405]

[0406] Screening of Oligoamines for the Release of GBS Capsular Polysaccharide from Cells

[0407] Hydroxylamine and its substituted compounds were found to be very effective in lysing capsular polysaccharides from the GBS cell wall. However, they were found to be less effective against serotypes II and V. Therefore, oligoamines were tested because they were believed to be more active due to having multiple amine functionalities.

[0408] Ethylene diamine was found to be effective in releasing capsular polysaccharides from all serotypes. The data are shown in Table 6 ([ethylenediamine] - 50 or 100 mM; pH 8.0; 16 h; 80 °C; 25 mM EDTA).

[0409] Table 6: GBS CP release data using ethylenediamine

[0410] GBS serotype Recovery (%) Mw (kDa) Ia 96％ 242 Ib 83％ 225 II 30％ 76 III 68％ 94 V 30％ 235

[0411] The activities of other representative oligoamines were tested using serotype Ia and V cell pastes, but they were also found to be less effective against serotype V. The data are shown in Table 7 ([triethylenetetramine] - 100 mM; pH - 8.9; temperature - 60 °C; time - 15 h), Table 8 ([1,1,4,7,10,10 - hexamethyltriethylenetetramine] - 10 mM; pH - 6.3; temperature - 60 °C; time - 20 h), and Table 9 ([2,6,10 - trimethyl - 2,6,10 - triazoundecane] - 10 mM; pH - 7 to 8; temperature - 60 °C; time - 19 h).

[0412] Table 7. GBS CP release data using triethylenetetramine

[0413]

[0414] GBS type Ia V Polysaccharide release yield % 100 After 2.5 days to 10 Molecular weight (Mw) 1280 nd

[0415] nd – not determined

[0416] Table 8. GBS CP release data using 1,1,4,7,10,10 - hexamethyltriethylenetetramine

[0417]

[0418] GBS type Ia V Polysaccharide release yield % 100 ～1％ Molecular weight (Mw) 980 nd

[0419] nd – not determined

[0420] Table 9. GBS CP release data using 2,6,10 - trimethyl - 2,6,10 - triazoundecane

[0421]

[0422] GBS type Ia V Polysaccharide release yield % 100 ～1％ Molecular weight (Mw) 1100 nd

[0423] nd – Not determined

[0424] Example 3. Conjugation of GBS capsular polysaccharide by reductive amination

[0425] Activating Polysaccharide

[0426] The polysaccharide oxidation reaction is carried out in 100 mM potassium phosphate buffer (pH 6.0 ± 0.5) to produce a polysaccharide with a final concentration of 2.0 g / L by sequentially adding a calculated amount of 500 mM potassium phosphate buffer (pH 6.0) and water for injection (WFI). If necessary, the reaction pH is adjusted to about pH 6.0. After adjusting the pH, the reaction temperature is adjusted to 23 °C. The oxidation reaction is initiated by adding about 0.25 molar equivalents of sodium periodate. The oxidation reaction is carried out at 5 ± 3 °C for about 16 hours.

[0427] The activated polysaccharide is concentrated and diafiltered using a 5K MWCO ultrafiltration cassette. Diafiltration is carried out against 20 times the diavolume of water for injection (WFI). Then, the purified activated polysaccharide is stored at 5 ± 3 °C. The characterization of the purified activated polysaccharide can be carried out, among other things, by (i) colorimetric assay to determine the sugar concentration; (ii) colorimetric assay to determine the aldehyde concentration; (iii) degree of oxidation; and (iv) determination of the molecular weight by SEC - MALLS.

[0428] The degree of oxidation of the activated polysaccharide (DO = moles of sugar repeat units / moles of aldehyde) is determined as follows:

[0429] The moles of sugar repeat units are determined by various colorimetric methods. For example, for the anthrone method, the polysaccharide is first decomposed into monosaccharides by the action of sulfuric acid and heat. The anthrone reagent reacts with hexose to form a yellow - green mixture, and its absorbance is read spectrophotometrically at 625 nm. In the analysis range, the absorbance is proportional to the amount of hexose present.

[0430] The molar number of aldehydes was also determined simultaneously using the MBTH colorimetric method. The MBTH analysis involves reacting the aldehyde group (from a given sample) with 3-methyl-2-benzothiazolone hydrazone (MBTH analysis reagent) to form an azine compound. The excess 3-methyl-2-benzothiazolone hydrazone is oxidized to form a reactive cation. The reactive cation reacts with the azine to form a blue chromophore. The formed chromophore is then read spectrophotometrically at 650 nm.

[0431] Mix the activated polysaccharide with the sucrose excipient and lyophilize.

[0432] The activated polysaccharide was mixed with sucrose at a ratio of 25 grams of sucrose per gram of activated polysaccharide. Then the bottle of the mixed mixture was lyophilized. After lyophilization, the bottle containing the lyophilized activated polysaccharide was stored at -20 ± 5 °C. The calculated amount of CRM 197 proteins were shell-frozen and lyophilized separately. The lyophilized CRM 197 was stored at -20 ± 5 °C.

[0433] Reconstitute the lyophilized activated polysaccharide and carrier protein

[0434] The lyophilized activated polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). When the polysaccharide was completely dissolved, an equal volume of anhydrous DMSO was added to the lyophilized CRM 197 for reconstitution.

[0435] Conjugating and Capping

[0436] The reconstituted activated polysaccharide was combined with the reconstituted CRM 197 in a reaction vessel and then thoroughly mixed to obtain a clear solution before initiating conjugation using sodium cyanoborohydride. The final polysaccharide concentration in the reaction solution was approximately 1 g / L. The conjugation reaction was initiated by adding 1.0 to 1.5 MEq of sodium cyanoborohydride to the reaction mixture and incubating at 23 ± 2 °C for 20 to 48 hours. The conjugation reaction was terminated by capping the unreacted aldehydes by adding 2 MEq of sodium borohydride (NaBH 4 ) and allowing the capping reaction to proceed at 23 ± 2 °C for 3 ± 1 hours.

[0437] Purify the conjugate

[0438] When preparing for purification by tangential flow filtration using a 100 - 300K MWCO membrane, the conjugate solution was diluted 1:10 with chilled 5 mM succinate - 0.9% saline (pH 6.0).

[0439] The diluted conjugate solution was passed through a 5 - μm filter and diafiltered using 5 mM succinate / 0.9% saline (pH 6.0) as the medium. After diafiltration was completed, the conjugate retentate was transferred through a 0.22 - μm filter. The conjugate was further diluted to a target sugar concentration of approximately 0.5 mg / mL with 5 mM succinate / 0.9% saline (pH 6). Alternatively, 20 mM histidine - 0.9% saline (pH 6.5) was used to purify the conjugate by tangential flow filtration using a 100 to 300K MWCO membrane. The final 0.22 - μm filtration step was completed to obtain the immunogenic conjugate.

[0440] Example 4: Effect of Altered Conjugation Conditions on GBS Polysaccharide - CRM 197 Effect on Conjugates

[0441] GBS serotype Ia, Ib, II, III, IV, and V conjugates were generated by deliberately altering periodate oxidation / reductive amination chemistry (PO / RAC) conditions, including the solvent for the reagents (aqueous medium versus DMSO), altering the sialic acid level and degree of oxidation / glyco - epitope modification in the initial polysaccharide. Generally, conjugates made using DMSO as the solvent were found to have lower levels of unreacted (free) polysaccharide, higher conjugate molecular weights, and higher sugar / protein ratios compared to conjugates made using an aqueous medium.

[0442] A conjugation method that produces conjugates with lower levels of unreacted (free) polysaccharide is advantageous and preferred. It is well known that high levels of unreacted (free) polysaccharide can cause excessive T - cell independent immune responses, which may dilute the T - cell dependent response generated by the polysaccharide - protein conjugate, thereby reducing the immunogenic response due to the conjugate.

[0443] The selected GBS polysaccharide is chemically desialylated by methods known in the art (see Chaffin, D.O, et al., J Bacteriol 187(13):4615-4626(2005)) to generate conjugate variants to determine the effect of the degree of desialylation % on immunogenicity. Desialylation exceeding about 40% (i.e., sialic acid levels less than about 60%) has a negative impact on immunogenicity.

[0444] Similarly, in most cases, an oxidation degree lower than about 5, or a saccharide epitope modification exceeding about 20% has a negative impact on immunogenicity. Since the oxidation reaction occurs through sialic acid on the capsular polysaccharide, the results seem to indicate that a saccharide epitope modification exceeding about 20% reduces the sialic acid content, which leads to a decrease in immunogenicity.

[0445] Conversely, conjugates with various sugar / protein ratios or polysaccharide molecular weights can generate immunogenic responses in mice, indicating a rather wide range of acceptance criteria for these properties.

[0446] Alternative chemical routes are also used to generate additional conjugate variants. One alternative chemistry involves reacting the polysaccharide with carbonylditriazole (CDT) and performing the conjugation reaction in DMSO to generate the conjugate. In another alternative chemistry, the polysaccharide is oxidized by using TEMPO [(2,2,6,6-tetramethylpiperidin-1-yl)oxyl] reagent (instead of sodium periodate), and then conjugated using reductive amination chemistry (TEMPO / RAC) in DMSO to generate the conjugate, as detailed in Example 3 above. All conjugates generated by these alternative chemistries were shown to be immunogenic in mice, indicating the suitability of alternative chemical routes other than PO / RAC. However, some conjugation chemistries perform better with certain serotypes than with others.

[0447] OPA was performed according to Nanra, J.S., et al., Hum. Vaccin. Immunother., 9(3):480 - 487(2013), but with group B streptococcal isolate instead of Staphylococcus aureus isolate, and the preopsonization step was omitted. The OPA titer after three doses (PD3) was provided as the geometric mean from a group of 10 to 20 mice immunized with 1 mcg / ml in each dose of the respective conjugate.

[0448] GBS serotype Ia polysaccharide - CRM 197 conjugate

[0449] Conjugates generated using PO / RAC and activated polysaccharides with DO of 16 to 17 (approx. 6% sugar epitope modification) were shown to be immunogenic (conjugates 1 and 3). However, using activated polysaccharides with DO of 5.4 (approx. 19% sugar epitope modification) had a negative impact on immunogenicity (conjugate 2). Similarly, a sialic acid level of 50% produced little immunogenic response (conjugate 4). The results are shown in Table 10.

[0450] Table 10. Effect of varying the conditions of periodate oxidation / reductive amination chemistry on GBS serotype Ia - CRM 197 conjugates

[0451]

[0452]

[0453] Additional conjugate variants were generated using alternative conjugation chemistries and conjugate molecular properties (results are shown in Table 11). Conjugate 5 was generated by reacting a polysaccharide with carbonylditriazole (CDT), and the conjugation reaction was carried out in DMSO. Conjugate 6 was generated by oxidizing the polysaccharide using a TEMPO reagent (instead of sodium periodate), followed by conjugation using reductive amination chemistry in DMSO, as detailed in Example 3 above. Conjugate 7 was generated by PO / RAC and deliberately varying the conjugation parameters to produce a conjugate with a high sugar / protein ratio (SPR). Conjugate 8 was generated by PO / RAC using a polysaccharide with a low MW (40 kDa). All of these conjugates were shown to be immunogenic in mice, indicating the suitability of alternative conjugation chemistries and alternative conjugate attributes (such as the SPR and low MW of the starting polysaccharide) in addition to periodate oxidation / reductive amination chemistry.

[0454] Table 11. Effect of Method Conditions for Altering Periodate Oxidation / Reductive Amination Chemistry and Alternative Chemistry Selection on GBS Serotype Ia-CRM 197 Conjugates

[0455]

[0456]

[0457] GBS Serotype Ib Polysaccharide-CRM 197 Conjugates

[0458] Conjugates generated using PO / RAC and activated polysaccharides with a DO of 15.8 (approx. 6% sugar epitope modification) in DMSO were shown to be immunogenic in mice (Conjugates 9 and 11). When all other conjugate molecular properties were similar (Conjugates 9 and 11, respectively), conjugates generated by PO / RAC in DMSO were slightly more immunogenic than those generated by PO / RAC in aqueous media. However, using activated polysaccharides with a DO of 4.7 (approx. 21% sugar epitope modification) had a negative impact on immunogenicity (Conjugate 10). In conjugates generated using PO / RAC and 95% desialylated (5% sialic acid level) polysaccharides (Conjugate 12), immunogenicity was almost completely abolished, with only a very few responders. Results are shown in Table 12.

[0459] Table 12. Effects of Method Conditions for Altering Periodate Oxidation / Reductive Amination Chemistry on GBS Serotype Ib-CRM 197 Conjugates

[0460]

[0461]

[0462] Additional conjugate variants were generated using alternative conjugation chemistries and conjugate molecular properties (results are shown in Table 13). Conjugate 13 was generated by PO / RAC using a polysaccharide with low sialylation (65%) in the initial polysaccharide. Conjugate 14 was generated by PO / RAC and deliberately altering the conjugation parameters to produce a conjugate with a high saccharide / protein ratio (SPR). Conjugate 15 was generated by reacting the polysaccharide with carbonylditriazole (CDT) and performing the conjugation reaction in DMSO. Conjugate 16 was generated by oxidizing the polysaccharide using a TEMPO reagent (instead of sodium periodate) followed by conjugation using reductive amination chemistry (TEMPO / RAC) in DMSO, as detailed in Example 3 above. All of these conjugates were shown to be immunogenic in mice, indicating the suitability of alternative conjugation chemistries in addition to periodate oxidation / reductive amination chemistry and alternative conjugate properties such as the SPR and low MW of the initial polysaccharide.

[0463] Table 13. Effects of Method Conditions for Altering Periodate Oxidation / Reductive Amination Chemistry and Alternative Chemistry Selection on GBS Serotype Ib-CRM 197 Conjugates

[0464]

[0465] GBS Serotype II Polysaccharide-CRM 197 Conjugates

[0466] Conjugates generated using PO / RAC and activated polysaccharides with a DO of 4 to 15 (about 7 to 23% sugar epitope modification) were shown to be immunogenic in mice (Conjugates 17 to 20). Conjugates generated using PO / RAC and a polysaccharide with a sialylation level of 74% (26% desialylation) were also shown to be immunogenic (Conjugate 20). Results are shown in Table 14.

[0467] Table 14. Effects of Method Conditions for Altering Periodate Oxidation / Reductive Amination Chemistry on GBS Serotype II-CRM 197 Conjugates

[0468]

[0469] Additional conjugate variants were generated using alternative conjugation chemistries and conjugate molecular properties (results are shown in Table 15). Conjugates 21 and 22 were generated by PO / RAC and deliberately varying the conjugation parameters to produce conjugates with low and high sugar / protein ratios (SPR), respectively. Conjugate 23 was generated by oxidizing 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 generated by reacting the polysaccharide with carbonylditriazole (CDT) and performing the conjugation reaction in DMSO. All of these conjugates were shown to be immunogenic in mice, indicating the suitability of alternative conjugation chemistries and alternative conjugate attributes such as SPR in addition to periodate oxidation / reductive amination chemistry.

[0470] Table 15. Effect of method conditions altering periodate oxidation / reductive amination chemistry and alternative chemistry choices on GBS serotype II-CRM 197 conjugates

[0471] GBS serotype III polysaccharide-CRM 197 conjugates

[0472] Conjugates generated using PO / RAC in DMSO and activated polysaccharides with a DO of 10 to 17 (approx. 6 to 10% sugar epitope modification) were shown to be immunogenic in mice (conjugates 25 and 30). Conjugates with a DO of 2.9 (approx. 34% sugar epitope modification) or a high sugar / protein ratio (2.1) (conjugates 26 and 27, respectively) showed relatively low immunogenicity. Conjugates generated using PO / RAC and polysaccharides with a sialylation level of 81% (19% desialylated) were shown to be immunogenic (conjugate 30). However, conjugates generated using polysaccharides with a sialylation level of 58% (42% desialylated) showed poor immunogenicity (conjugate 29). When the properties of all other conjugate molecules were similar (conjugates 25 and 28, respectively), conjugates generated by PO / RAC in DMSO were slightly more immunogenic than those generated by PO / RAC in aqueous media. Results are shown in Table 16.

[0473] Table 16. Effect of method conditions altering periodate oxidation / reductive amination chemistry on GBS serotype III-CRM 197 conjugates

[0474]

[0475]

[0476] Additional conjugate variants were generated using alternative conjugation chemistries and conjugate molecular properties (results shown in Table 17). Conjugates 31 to 35 were generated by PO / RAC and deliberately varying the conjugation parameters to produce conjugates with different MWs. Conjugate 36 was generated by reacting the polysaccharide with carbonylditriazole (CDT) and performing the conjugation reaction in DMSO. Conjugate 37 was generated by oxidizing 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 of these conjugates were demonstrated to be immunogenic in mice, indicating the suitability of alternative conjugation chemistries and alternative conjugate properties (such as MW) in addition to the periodate oxidation / reductive amination chemistry. Compared to the conjugate (conjugate 26 in Table 16 above) generated with a DO of 2.9 (approximately 34% sugar epitope modification), the conjugate generated with a DO as low as 5 (approximately 20% sugar epitope modification) was still immunogenic in mice (conjugate 32 in Table 17).

[0477] Table 17. Effect of Method Conditions for Altering Periodate Oxidation / Rductive Amination Chemistry and Alternative Chemistry Selection on GBS Serotype III-CRM 197 Conjugates

[0478]

[0479]

[0480] GBS Serotype IV Polysaccharide-CRM 197 Conjugates

[0481] Conjugates generated using PO / RAC and activated polysaccharides with DOs of 6.9 to 14.2 (approximately 7 to 14% saccharide epitope modification) were shown to be immunogenic in mice (conjugates 38 to 41). Conjugates generated using PO / AC and polysaccharides with 60% sialylation (40% desialylation) were also shown to be immunogenic (conjugate 41). Results are shown in Table 18.

[0482] Table 18. Effect of Method Conditions for Altering Periodate Oxidation / Rductive Amination Chemistry on GBS Serotype IV-CRM 197 Conjugates

[0483]

[0484]

[0485] Additional conjugate variants were generated using alternative conjugation chemistries and conjugate molecular properties. The results are shown in Table 19. Conjugates 42 and 45 were generated by PO / RAC and deliberately varying the conjugation parameters to produce conjugates with high DO (lower oxidation level) and high SPR, respectively. Conjugate 43 was generated by reacting the polysaccharide with carbonylditriazole (CDT) and performing the conjugation reaction in DMSO. Conjugate 44 was generated by oxidizing the polysaccharide using TEMPO reagent (instead of sodium periodate) and then conjugating it using reductive amination chemistry in DMSO, as detailed in Example 3 above. All serotype IV conjugates were shown to be immunogenic in mice, indicating the suitability of alternative conjugation chemistries other than periodate oxidation / reductive amination chemistry and conjugate properties such as SPR. Conjugates generated with a DO of up to at least 20 (lower oxidation) (approx. 5% sugar epitope modification) were still immunogenic in mice.

[0486] Table 19. Effect of method conditions for altering periodate oxidation / reductive amination chemistry and alternative chemistry selection on GBS serotype IV-CRM 197 conjugates

[0487]

[0488] GBS serotype V polysaccharide-CRM 197 conjugates

[0489] Conjugates generated using PO / RAC and activated polysaccharides with a DO of 4.4 to 14.6 (approx. 7 to 23% sugar epitope modification) were shown to be immunogenic in mice (conjugates 46 and 47). Desialylated conjugates (5% sialylation level) were not immunogenic (conjugate 49), while conjugates generated using an aqueous solvent with the PO / RAC method produced a weak immune response (conjugate 48). The results are shown in Table 20.

[0490] Table 20. Effect of method conditions for altering periodate oxidation / reductive amination chemistry on GBS serotype V-CRM 197 conjugates

[0491]

[0492] Additional conjugate variants were generated using alternative conjugation chemistries and conjugate molecular properties. The results are shown in Table 21. Conjugates 50 and 53 were generated by PO / RAC and deliberately varying the conjugation parameters to produce conjugates with lower degrees of sialylation (81% sialylation) and low molecular weight (MW), respectively. Conjugate 51 was generated by reacting the polysaccharide with carbonylditriazole (CDT) and performing the conjugation reaction in DMSO. Conjugate 52 was generated by oxidizing the polysaccharide using a TEMPO reagent (instead of sodium periodate) followed by conjugation using reductive amination chemistry in DMSO, as described in Example 3 above. All serotype V conjugates (except those generated using CDT chemistry) were shown to be immunogenic in mice, indicating the suitability of alternative conjugation chemistries in addition to periodate oxidation / reductive amination chemistry and conjugate properties such as MW. The conjugate generated using CDT chemistry showed significantly weaker immunogenicity compared to other conjugates generated by RAC. The 81% sialylated conjugate (conjugate 50) provided a weaker immune response compared to the >95% sialylated conjugate (conjugate 53), but a stronger response compared to the 5% sialylated conjugate (conjugate 49 in Table 20 above).

[0493] Table 21. Effect of modifying method conditions of periodate oxidation / reductive amination chemistry and alternative chemistry selection on GBS serotype V-CRM 197 conjugates

[0494] Example 5: GBS III-CRM 197 and GBS V-CRM 197 monovalent conjugate vaccines elicit an OPA response in mice

[0495] Female CD-1 mice were immunized three times via the subcutaneous route at 0, 3, and 6 weeks with 1 mcg, 0.1 mcg, or 0.01 mcg of Streptococcus agalactiae (GBS) serotype III conjugated to CRM 197 (GBS III-CRM 197 ), or GBS serotype V conjugated to CRM 197 (GBS V-CRM 197 ). Serum was evaluated at post dose three (PD3) by opsonophagocytic assay (OPA). OPA was performed as described in Example 4. Both conjugates elicited an OPA response in mice (Table 22). Samples with no detectable OPA response were assigned a value of 50.

[0496] Table 22: OPA responses elicited by GBS III and GBS V conjugates in mice

[0497]

[0498] Example 6: GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBSⅡ-CRM 197 , GBSⅢ-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 Monovalent conjugate vaccines produce OPA responses in mice

[0499] Six groups of female CD-1 mice were immunized three times via the subcutaneous route at weeks 0, 3, and 6 with a vaccine containing 1 mcg of individual GBS capsular polysaccharide (CP) conjugated to CRM 197 . A preliminary study showed that the mice did not have pre-existing OPA titers to any of the six serotypes tested. Sera from PD3 were analyzed for 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.

[0500] Table 23: Geometric mean OPA titers of mice immunized with individual GBS CPS-CRM 197 conjugates

[0501]

[0502] Table 24: Fold-rise OPA titers of mice immunized with individual GBS CPS-CRM 197 conjugates

[0503]

[0504] Note: Fold-rise calculations assume that the mice did not have pre-existing titers.

[0505] Example 7: Serum opsonic activity compared to IgG isolated from mice immunized with monovalent conjugate vaccines of GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBSⅡ-CRM 197 , GBSⅢ-CRM 197 , GBSIV-CRM 197 and GBS V-CRM 197 ​

[0506] At weeks 0, 3 and 6, female CD-1 or BALB / c mice were immunized three times via the subcutaneous route with 1 mcg of individual GBS CPs conjugated to CRM 197 using AlPO 4 or QS-21 as adjuvant. PD3 sera were tested by serotype-specific OPA, then the immunoglobulin G fraction was 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 sera). All six GBS CPS conjugates induced IgG antibodies with opsonic activity( Figure 1 ).

[0507] Example 8: Monovalent conjugate vaccines of GBS Ia-TT, GBS Ib-TT, GBS II-TT, GBS III-TT, GBS IV-TT and GBS V-TT elicit OPA responses in rabbits

[0508] Rabbits were immunized three times with 50 mcg / ml of GBS serotype Ia polysaccharide conjugated to tetanus toxoid, 10 mcg / ml of GBS serotype Ib polysaccharide conjugated to tetanus toxoid, 50 mcg / ml of GBS serotype II polysaccharide conjugated to tetanus toxoid, 50 mcg / ml of GBS serotype III polysaccharide conjugated to tetanus toxoid, 50 mcg / ml of GBS serotype IV polysaccharide conjugated to tetanus toxoid, or 50 mcg / ml of GBS serotype V polysaccharide conjugated to tetanus toxoid, with complete Freund's adjuvant in the first dose and incomplete Freund's adjuvant in the second and third doses. The conjugate was manufactured using polysaccharide with sialic acid levels > 95% and CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate). PD3 immune responses were measured by OPA as described in Example 4. Serum titers are shown in Table 25, and purified IgG titers are shown in Table 26 below. GBS serotype Ia, Ib, II, III, IV and V polysaccharides conjugated to TT are highly immunogenic in rabbits.

[0509] Table 25: Geometric mean OPA titers of rabbit sera after immunization with individual GBS CPS-TT conjugates

[0510]

[0511] Table 26: Geometric mean OPA titers of rabbit sera after immunization with individual GBS CPS-TT conjugates

[0512]

[0513] Example 9: Hexavalent GBS Conjugate Vaccine Elicits an OPA Response in Non-Human Primates

[0514] Three groups of rhesus macaques were immunized three times at weeks 0, 4, and 8 via the intramuscular route with a hexavalent group B streptococcus (GBS6) vaccine. The GBS6 vaccine included GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 , and GBS V-CRM 197 . Two groups included aluminum phosphate (AlPO 4 ) as an adjuvant and received either 5 mcg of each conjugate or 50 mcg of each conjugate. The third group received 5 mcg of each conjugate and no adjuvant. The following Table 27 describes the immunization schedule.

[0515] Table 27: Immunization schedule of rhesus macaques

[0516]

[0517]

[0518] All six GBS serotypes included in the vaccine were analyzed by OPA in preimmune sera and sera from PD3. OPA was performed as described in Example 4. The results are shown in Tables 28 and 29 below. For all six serotypes, formulations with aluminum phosphate (AlPO 4 ) adjuvant elicited a detectable OPA response (increase in titer from pre-immune (pre) to PD3; or fold increase pre / PD3 > 1). The 5 mcg / conjugate dose without adjuvant elicited a detectable OPA response for 5 out of 6 serotypes.

[0519] Table 28: Geometric mean OPA titers of rhesus macaques before and after immunization with GBS6

[0520]

[0521] Table 29: Fold Rise in OPA Titers in Rhesus Monkeys after Immunization with GBS6

[0522]

[0523] Example 10: Hexavalent GBS Conjugate Vaccine Elicits an OPA Response in Rats

[0524] Female Sprague-Dawley rats were immunized twice via the subcutaneous route with each conjugate in a GBS6 polysaccharide conjugate vaccine (formulated as in Example 9, with or without aluminum phosphate (AlPO 4 )) at 5 mcg / ml. OPA assay titers of two sera preimmune (baseline) and post-dose were evaluated against all six cognate GBS serotypes. OPA titers for each serotype were measured in a GBS 384-well assay format and fold rise was calculated. Rats administered the GBS6 vaccine had a robust functional antibody response to each serotype after the second dose; when no AlPO 4 was present, a 7- to 205-fold increase was seen between serotypes, while in the presence of AlPO 4 , this ranged from 11 to 294-fold (Table 30). Table 30. Fold Rise in Titers after the Second Dose (PD2) in Rats Immunized with Hexavalent GBS Conjugate Vaccine in an Opsonophagocytic Activity (OPA) Assay

[0525]

[0526] Example 11: Pregnant Dams Immunized with Monovalent or Hexavalent GBS Glycoconjugate Vaccines Show Protection of Their Offspring from GBS Type III or V Infection after Birth

[0527] Via the subcutaneous route with GBS6 vaccine, GBS type III or V monovalent glycoconjugate vaccines (each containing 10 mcg / ml of conjugate and 100 mcg / ml of AlPO 4 ), as described in Example 9, containing 5 mcg / ml of each conjugate and 100 mcg / ml of AlPO 4) or a separate vehicle control group immunized female CD-1 mice three times. Breed the mice before the third immunization. Challenge the offspring of the immunized mice with a lethal dose of GBS serotype III or GBS serotype V bacteria according to the vaccine received, and monitor survival for 90 hours. With GBS6 + AlPO 4 or GBS III-CRM 197 + AlPO 4 Immunized dams provided significant protection (p < 0.0001) to their pups against a lethal GBS serotype III challenge. Similarly, with GBS V-CRM 197 + AlPO 4 Immunized dams provided significant protection (p < 0.0001) to their pups against a lethal GBS serotype V challenge. The results are shown in Table 31.

[0528] Table 31: Immunization with monovalent and hexavalent GBS vaccines increases offspring survival

[0529]

[0530] Example 12: Passive Immunization with GBS III Monoclonal Antibody in Pups Shows Protective Effect

[0531] Immunize mice with a pentavalent vaccine including serotypes Ia, Ib, II, III, and V to generate group B streptococcus serotype III (GBS III) monoclonal antibody (mAb). Select GBS III-specific mAb clones and use standard procedures to generate mAbs that recognize the CP of GBS III. Sixteen hours before challenge with a clinical GBS III isolate, passively administer GBS serotype III mAb to pups (n = 10 per group; 2 independent experiments shown). Blood is collected four hours after challenge and the remaining CFU is calculated. Treatment with GBS III mAb can reduce the recovered CFU in pups by 4 log or more (see Table 32).

[0532] Table 32: GBS III mAb Reduces Recovered CFU in Pups

[0533]

[0534]

[0535] Example 13: Passive immunization with GBS Ib, III, and V monoclonal antibodies in pregnant mice shows protective effects on their offspring after birth

[0536] Monoclonal antibodies (mAbs) were generated from mice immunized with a pentavalent vaccine (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBSIII-CRM 197 , and GBS V-CRM 197 ) using standard procedures. The mAbs were then confirmed to specifically recognize the capsular polysaccharides of each of the 5 serotypes. Approximately 24 to 48 hours before parturition of pregnant mice, a dose of 500 mcg / ml of GBS serotype Ib (GBS Ib) mAb, GBS serotype III (GBS III) mAb, GBS serotype V (GBS V) mAb, or isotype-matched control mAb was passively administered to the pregnant mice. Twenty-four to 48 hours after birth, the offspring of the immunized dams were challenged with a lethal dose of GBS Ib, GBS III, or GBS V bacteria. Survival was monitored for 96 hours. The pups born to dams immunized with GBS Ib mAb, GBS III mAb, or GBS V mAb had significantly higher survival rates after GBS challenge compared to those immunized with control mAb (see Table 33).

[0537] Table 33: GBS III&V mAb increases offspring survival

[0538]

[0539] Example 14: Stability of GBS conjugates

[0540] GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 , and GBS V-CRM 197, to test the stability of the conjugate under accelerated storage conditions. The percentage change in molecular weight (determined by SEC MALLS) was measured after storage at 50 °C for 4 weeks. The results are shown in Figures 2 to 7 .

[0541] The sialylation stability of GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS III-CRM 197 and GBS IV-CRM 197 conjugates was tested under various buffer conditions shown in Table 34. After storage at 37 °C for 1 month, free sialic acid (N-acetylneuraminic acid; NANA) was measured using HPLC. The results are shown in Figure 8.

[0542] Two studies showed that the conjugate performed better above pH 6.0 and most ideally at approximately pH 6.5.

[0543] Table 34. Buffer conditions for sialylation stability tests

[0544]

[0545] Example 15: GBS6 formulation

[0546] To determine the buffer selection, GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM 197 , GBS IV-CRM 197 and GBS V-CRM 197 (GBS6) were formulated together using the same buffer conditions as shown in Table 33 above. The actual pH of such formulations was tested at the following time points: 0 (when formulating the preparation), after 1 month at 5 °C, after 1 month at 25 °C, and after 1 month at 37 °C. A pH shift was seen in the formulation using succinate as the buffer, while no shift was seen in the formulation using histidine as the buffer. The results are shown in Figures 9 to 10 .

[0547] The effect of histidine buffer concentration on the binding of GBS conjugates to aluminum was also tested. Using two different concentrations of the conjugate (10 mcg / ml and 40 mg / ml of each serotype) and several different concentrations of histidine, including 150 mM NaCl, 0.01% polysorbate-80 at pH 6.5, and 0.5 mg / ml of AlPO4 an aluminum formulation. The percentage of conjugate bound to aluminum is determined by measuring the total amount of each conjugate in the vaccine and the amount of each conjugate bound to aluminum. The bound conjugate is measured by the following steps: centrifuging the vaccine formulation, resuspending the aluminum precipitate, dissolving the aluminum, and measuring the bound conjugate using nephelometry with serotype-specific polyclonal antibodies against each serotype. The results are shown in Figures 11 to 12 . It was found that the concentration of the histidine buffer affects the percentage of each serotype bound to aluminum, and this effect is more pronounced at lower doses than at higher doses.

[0548] An agitation study was conducted to determine the ideal amount of polysorbate-80 (PS80). Test the total antigenicity lost percentage of GBS6 formulations containing 20 mM histidine, 150 mM NaCl, 0.5 mg / ml AlPO 4 (if present), and without PS80, 0.01% PS80, 0.02% PS80, or 0.03% PS80 (pH 6.5) under agitation stress. Syringes pre-filled with the formulation were agitated at 500 RPM at room temperature for 72 hours. Control samples (not agitated) were stored at room temperature for 72 hours. The results are shown in Figure 13 .

[0549] The aluminum concentration in the GBS6 formulation was also studied to determine its effect on the binding of the GBS conjugate to aluminum. Test the percentage of conjugate bound to aluminum for GBS6 formulations containing 10 mM histidine, 150 mM NaCl, 0.02% PS80, and 0.25 mg / ml, 0.5 mg / ml, or 0.75 mg / ml, or 1.0 mg / ml of aluminum as aluminum phosphate (AlPO 4 ). The percentage bound to AlPO 4 increases with increasing AlPO 4 concentration. The results are shown in Figure 14 .

[0550] Example 16: GBS6 Lyophilized Formulation

[0551] Test GBS6 (GBS Ia-CRM 197 , GBS Ib-CRM 197 , GBS II-CRM 197 , GBS III-CRM197 , GBS IV-CRM 197 and GBS V-CRM 197 ) of various lyophilized formulations. Low (10 mcg / ml) and high (50 mcg / ml) dose formulations containing 20 mM histidine (pH 6.5), 0.02% PS80, approximately 28 mM NaCl, and 5.5%, 7.0%, or 8.5% (w / v) sucrose were lyophilized. After 4 months at 5°C, 4 months at 37°C, and 1 month at 50°C, pH and moisture were measured to test the stability of the lyophilized formulations. Based on pH and moisture (data not shown), all formulations were stable. In addition, the percentage of antigenicity recovery of each serotype was tested for all formulations after 1, 4, and 9 months at 5°C and 37°C and after 1, 2, and 4 weeks at 50°C. The results are shown in Figures 15 to 20 .

[0552] The following changes to the excipients of the GBS6 formulation at a dose of 40 mcg / ml were also prepared and evaluated: 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. The pH and moisture of all five formulations were 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 antigenicity recovery of 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 .

[0553] The percentage of antigen bound to aluminum phosphate adjuvant in the reconstituted lyophilized formulation and liquid formulation of the GBS6 vaccine was tested using turbidimetry. Low (10 mcg / ml) and high (50 mcg / ml) dose lyophilized and liquid formulations containing 20 mM histidine, 0.02% PS80, 7.0% (w / v) sucrose, and 500 mcg / ml of aluminum as aluminum phosphate were prepared. The effect of altered sodium chloride (NaCl) concentration on antigen binding was also tested. The results for the lyophilized and liquid formulations are shown in Tables 35 and 36, respectively. When using an NaCl concentration of approximately 150 mM or higher, the low dose formulations of both the lyophilized and liquid compositions had comparable results.

[0554] Table 35. Percentage of antigen bound to aluminum phosphate in formulated lyophilized formulations with different amounts of NaCl

[0555]

[0556]

[0557] Table 36. Percentage of antigen bound to aluminum phosphate in liquid formulations with different amounts of NaCl

[0558]

[0559] Aspects of the invention

[0560] The following describe additional embodiments of the invention:

[0561] C1. An immunogenic polysaccharide-protein conjugate comprising a Streptococcus agalactiae (GBS) capsular polysaccharide and a carrier protein, wherein the sialic acid level of the capsular polysaccharide is greater than about 60%.

[0562] C2. The 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.

[0563] C3. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype Ia.

[0564] C4. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype Ib.

[0565] C5. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype II.

[0566] C6. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype III.

[0567] C7. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype IV.

[0568] C8. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype V.

[0569] C9. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype VI.

[0570] C10. The immunogenic conjugate of C2, wherein the capsular polysaccharide is serotype VII.

[0571] C11. An immunogenic conjugate as in C2, wherein the capsular polysaccharide is serotype VIII.

[0572] C12. An immunogenic conjugate as in C2, wherein the capsular polysaccharide is serotype IX.

[0573] C13. An immunogenic conjugate as in any one of C1 to C12, wherein the sialic acid level of the capsular polysaccharide is higher than about 95%.

[0574] C14. An immunogenic conjugate as in any one of C1 to C13, wherein the sialic acid level of the capsular polysaccharide is about 100%.

[0575] C15. An immunogenic conjugate as in any one of C1 to C14, wherein the capsular polysaccharide has at least about 0.6 mM of sialic acid per mM of polysaccharide.

[0576] C16. An immunogenic conjugate as in any one of C1 to C15, wherein the capsular polysaccharide has at least about 0.65 mM of sialic acid per mM of polysaccharide.

[0577] C17. An immunogenic conjugate as in any one of C1 to C16, wherein the capsular polysaccharide has at least about 0.7 mM of sialic acid per mM of polysaccharide.

[0578] C18. An immunogenic conjugate as in any one of C1 to C17, wherein the capsular polysaccharide has at least about 0.75 mM of sialic acid per mM of polysaccharide.

[0579] C19. An immunogenic conjugate as in any one of C1 to C18, wherein the capsular polysaccharide has at least about 0.8 mM of sialic acid per mM of polysaccharide.

[0580] C20. An immunogenic conjugate as in any one of C1 to C19, wherein the capsular polysaccharide has at least about 0.85 mM of sialic acid per mM of polysaccharide.

[0581] C21. An immunogenic conjugate as in any one of C1 to C20, wherein the capsular polysaccharide has at least about 0.9 mM of sialic acid per mM of polysaccharide.

[0582] C22. An immunogenic conjugate as in any one of C1 to C21, wherein the capsular polysaccharide has at least about 0.95 mM of sialic acid per mM of polysaccharide.

[0583] C23. An immunogenic conjugate as in any one of C1 to C22, wherein the molecular weight of the capsular polysaccharide is from about 5 kDa to about 1,000 kDa.

[0584] C24. An immunogenic conjugate as in any one of C1 to C23, wherein the molecular weight of the capsular polysaccharide is from about 25 kDa to about 750 kDa.

[0585] C25. An immunogenic conjugate as in any one of C1 to C24, wherein the molecular weight of the capsular polysaccharide is from about 25 kDa to about 400 kDa.

[0586] C26. An immunogenic conjugate as in any one of C1 to C25, wherein the molecular weight of the capsular polysaccharide is from about 25 kDa to about 200 kDa.

[0587] C27. An immunogenic conjugate as in any one of C1 to C25, wherein the molecular weight of the capsular polysaccharide is from about 100 kDa to about 400 kDa.

[0588] C28. An immunogenic conjugate as in any one of C1 to C27, wherein the molecular weight of the conjugate is from about 300 kDa to about 20,000 kDa.

[0589] C29. An immunogenic conjugate as in any one of C1 to C28, wherein the molecular weight of the conjugate is from about 1,000 kDa to about 15,000 kDa.

[0590] C30. An immunogenic conjugate as in any one of C1 to C29, wherein the molecular weight of the conjugate is from about 1,000 kDa to about 10,000 kDa.

[0591] C31. An immunogenic conjugate as in any one of C1 to C30, wherein the capsular polysaccharide is from about 0% to about 40% O-acetylated.

[0592] C32. An immunogenic conjugate as in any one of C1 to C31, wherein the O-acetylation of the capsular polysaccharide is less than about 5%.

[0593] C33. An immunogenic conjugate as in any one of C1 to C32, wherein the O-acetylation of the capsular polysaccharide is less than about 4%.

[0594] C34. An immunogenic conjugate as in any one of C1 to C33, wherein the O-acetylation of the capsular polysaccharide is less than about 3%.

[0595] C35. An immunogenic conjugate as in any one of C1 to C34, wherein the O-acetylation of the capsular polysaccharide is less than about 2%.

[0596] C36. An immunogenic conjugate as in any one of C1 to C35, wherein the O-acetylation of the capsular polysaccharide is less than about 1%.

[0597] C37. An immunogenic conjugate as in any one of C1 to C36, wherein the capsular polysaccharide comprises at least about 0.1 mM O - acetate per mM sugar repeat unit.

[0598] C38. An immunogenic conjugate as in any one of C1 to C37, wherein the capsular polysaccharide comprises at least about 0.2 mM O - acetate per mM sugar repeat unit.

[0599] C39. An immunogenic conjugate as in any one of C1 to C38, wherein the capsular polysaccharide comprises at least about 0.3 mM O - acetate per mM sugar repeat unit.

[0600] C40. An immunogenic conjugate as in any one of C1 to C39, wherein the capsular polysaccharide comprises at least about 0.35 mM O - acetate per mM sugar repeat unit.

[0601] C41. An immunogenic conjugate as in any one of C1 to C40, wherein the capsular polysaccharide comprises about 0.4 mM O - acetate per mM sugar repeat unit.

[0602] C42. An immunogenic conjugate as in any one of C1 to C41, wherein the capsular polysaccharide comprises less than about 0.01 mM O - acetate per mM sugar repeat unit.

[0603] C43. An immunogenic conjugate as in any one of C1 to C42, wherein the capsular polysaccharide comprises less than about 0.05 mM O - acetate per mM sugar repeat unit.

[0604] C44. An immunogenic conjugate as in any one of C1 to C43, wherein the capsular polysaccharide comprises less than about 0.04 mM O - acetate per mM sugar repeat unit.

[0605] C45. An immunogenic conjugate as in any one of C1 to C44, wherein the capsular polysaccharide comprises less than about 0.03 mM O - acetate per mM sugar repeat unit.

[0606] C46. An immunogenic conjugate as in any one of C1 to C45, wherein the capsular polysaccharide comprises less than about 0.02 mM O - acetate per mM sugar repeat unit.

[0607] C47. An immunogenic conjugate as in any one of C1 to C46, wherein the polysaccharides are each independently conjugated to a carrier protein.

[0608] C48. An immunogenic conjugate as in any one of C1 to C47, wherein the carrier protein is CRM 197 or tetanus toxoid.

[0609] C49. An immunogenic conjugate as in any one of C1 to C48, wherein the carrier protein is CRM 197 .

[0610] C50. A method for isolating a capsular polysaccharide, which comprises reacting an organic reagent with a cell broth containing a bacterium producing the capsular polysaccharide.

[0611] C51. The method as in C50, wherein the bacterium is not lysed.

[0612] C52. The method as in C50 or 51, wherein the bacterium is heat killed.

[0613] C53. The method as in any one of C50 to 52, wherein the method further comprises a centrifugation step to provide a cell paste.

[0614] C54. The method as in any one of C50 to 53, wherein the method further comprises a filtration step.

[0615] C55. The method as in C54, wherein the filtration step is diafiltration.

[0616] C56. The method as in any one of C50 to 55, wherein the bacterium producing the capsular polysaccharide 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.

[0617] C57. The method as in C56, wherein the bacterium is Streptococcus agalactiae.

[0618] C58. The method as in any one of C50 to 57, wherein the organic reagent is a derivatized hydroxyl amine compound.

[0619] C59. The method according to any one of C50 to C58, wherein the hydroxylamine is any of the hydroxylamines listed in Table 2 of Example 2.

[0620] C60. The method according to any one of C50 to C59, wherein the hydroxylamine is selected from the group consisting of: dibenzyl hydroxylamine; diethyl hydroxylamine; hydroxylamine; ethylenediamine; triethylenetetramine; 1,1,4,7,10,10 - hexamethyl triethylene tetramine; and 2,6,10 - Trimethyl 2,6,10 - triazaundecane.

[0621] C61. The method according to any one of C50 to C60, wherein the concentration of the hydroxylamine is from about 5 mM to about 200 mM.

[0622] C62. The method according to any one of C50 to C61, wherein the pH of the reaction is from about 5.5 to about 9.5.

[0623] C63. The method according to any one of C50 to C62, wherein the reaction is carried out at a temperature of from about 20 °C to about 85 °C.

[0624] C64. The method according to any one of C50 to C63, wherein the reaction time is from about 10 hours to about 90 hours.

[0625] C65. A method for producing an immunogenic polysaccharide - protein conjugate according to any one of C1 to C49, wherein the capsular polysaccharide is isolated according to the method of any one of C50 to C64.

[0626] C66. An immunogenic polysaccharide - protein conjugate comprising a capsular polysaccharide produced by the method according to any one of C50 to C64.

[0627] C67. An immunogenic composition comprising an immunogenic polysaccharide - protein conjugate according to any one of C1 to C49 or C66.

[0628] C68. Immunogenic composition, which comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from Streptococcus agalactiae (GBS) serotype IV and at least one additional serotype, and the additional serotype is selected from the group consisting of: Ia, Ib, II, III, V, VI, VII, VIII, and IX.

[0629] C69. The immunogenic composition according to C68, wherein the at least one additional serotype is Ia.

[0630] C70. The immunogenic composition according to C69, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype Ib.

[0631] C71. The immunogenic composition according to C69 or C70, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype II.

[0632] C72. The immunogenic composition according to any one of C69 to C71, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype III.

[0633] C73. The immunogenic composition according to any one of C69 to C72, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype V.

[0634] C74. The immunogenic composition according to any one of C69 to C73, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype VI.

[0635] C75. The immunogenic composition according to any one of C69 to C74, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype VII.

[0636] C76. The immunogenic composition according to any one of C69 to C75, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype VIII.

[0637] C77. The immunogenic composition according to any one of C69 to C76, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype IX.

[0638] C78. The immunogenic composition according to C68, wherein the at least one additional serotype is Ib.

[0639] C79. The immunogenic composition according to C78, wherein the composition further comprises a conjugate containing capsular polysaccharide from GBS serotype II.

[0640] C80. An immunogenic composition as in C78 or C79, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype III.

[0641] C81. An immunogenic composition as in any one of C78 to C80, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype V.

[0642] C82. An immunogenic composition as in any one of C78 to C81, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype VI.

[0643] C83. An immunogenic composition as in any one of C78 to C82, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype VII.

[0644] C84. An immunogenic composition as in any one of C78 to C83, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype VIII.

[0645] C85. An immunogenic composition as in any one of C78 to C84, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype IX.

[0646] C86. An immunogenic composition as in C68, wherein the at least one additional serotype is II.

[0647] C87. An immunogenic composition as in C86, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype III.

[0648] C88. An immunogenic composition as in C86 or C87, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype V.

[0649] C89. An immunogenic composition as in any one of C86 to C88, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype VI.

[0650] C90. An immunogenic composition as in any one of C86 to C89, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype VII.

[0651] C91. An immunogenic composition as in any one of C86 to C90, wherein the composition further comprises a conjugate containing a capsular polysaccharide from GBS serotype VIII.

[0652] C92. An immunogenic composition according to any one of C86 to C91, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype IX.

[0653] C93. An immunogenic composition according to C68, wherein the at least one additional serotype is III.

[0654] C94. An immunogenic composition according to C93, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype V.

[0655] C95. An immunogenic composition according to C93 or C94, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VI.

[0656] C96. An immunogenic composition according to any one of C93 to C95, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VII.

[0657] C97. An immunogenic composition according to any one of C93 to C96, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VIII.

[0658] C98. An immunogenic composition according to any one of C93 to C97, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype IX.

[0659] C99. An immunogenic composition according to C68, wherein the at least one additional serotype is V.

[0660] C100. An immunogenic composition according to C99, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VI.

[0661] C101. An immunogenic composition according to any one of C99 or C100, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VII.

[0662] C102. An immunogenic composition according to any one of C99 to C101, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VIII.

[0663] C103. An immunogenic composition according to any one of C99 to C102, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype IX.

[0664] C104. An immunogenic composition according to C68, wherein the at least one additional serotype is VI.

[0665] C105. An immunogenic composition as in 104, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VII.

[0666] C106. An immunogenic composition as in any one of C104 or C105, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VIII.

[0667] C107. An immunogenic composition as in any one of C104 to C106, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype IX.

[0668] C108. An immunogenic composition as in C68, wherein the at least one additional serotype is VII.

[0669] C109. An immunogenic composition as in C108, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype VIII.

[0670] C110. An immunogenic composition as in any one of C108 or C109, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype IX.

[0671] C111. An immunogenic composition as in C68, wherein the at least one additional serotype is VIII.

[0672] C112. An immunogenic composition as in C111, wherein the composition further comprises a conjugate comprising a capsular polysaccharide from GBS serotype IX.

[0673] C113. An immunogenic composition as in C112, wherein the at least one additional serotype is IX.

[0674] C114. An immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from GBS serotypes Ia, Ib, II, III, and IV.

[0675] C115. An immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from GBS serotypes Ia, Ib, II, III, and V.

[0676] C116. An immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharides from GBS serotypes Ia, Ib, II, III, IV, and V.

[0677] C117. An immunogenic composition comprising a polysaccharide-protein conjugate, the 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.

[0678] C118. The immunogenic composition of C117, wherein the composition comprises at least five GBS capsular polysaccharide serotypes.

[0679] C119. The immunogenic composition of C117 or C118, wherein the composition comprises at least six GBS capsular polysaccharide serotypes.

[0680] C120. The immunogenic composition of any one of C117 to C119, wherein the composition comprises at least seven GBS capsular polysaccharide serotypes.

[0681] C121. The immunogenic composition of any one of C117 to C120, wherein the composition comprises at least eight GBS capsular polysaccharide serotypes.

[0682] C122. The immunogenic composition of any one of C117 to C121, wherein the composition comprises at least nine GBS capsular polysaccharide serotypes.

[0683] C123. The immunogenic composition of any one of C117 to C122, wherein the composition comprises the GBS capsular polysaccharide serotype V.

[0684] C124. The immunogenic composition of any one of C117 to C123, wherein the composition does not have immune interference.

[0685] C125. The immunogenic composition of any one of C67 to C124, wherein the composition further comprises a pharmaceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic detergent, free radical oxidation inhibitor, carrier, or a mixture thereof.

[0686] C126. The immunogenic composition of any one of C67 to C125, wherein the composition further comprises a buffer.

[0687] 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.

[0688] C128. An immunogenic composition as in C127, wherein the buffer is histidine.

[0689] C129. An immunogenic composition as in any one of C67 to C128, wherein the composition further comprises a surfactant.

[0690] C130. An immunogenic composition as in C129, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid ester, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20, and polyoxyethylene alkyl ether.

[0691] C131. An immunogenic composition as in C130, wherein the surfactant is polysorbate-80.

[0692] C132. An immunogenic composition as in any one of C67 to C131, wherein the composition further comprises an excipient.

[0693] C133. An immunogenic composition as in 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, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim milk powder, glycerol, propylene glycol, water, and ethanol.

[0694] C134. An immunogenic composition as in C133, wherein the excipient is sodium chloride.

[0695] C135. An immunogenic composition as in any one of C67 to C134, wherein the composition further comprises an adjuvant.

[0696] C136. An immunogenic composition as in C135, wherein the adjuvant is an aluminum-based adjuvant or QS-21.

[0697] C137. An immunogenic composition as in C136, wherein the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyl phosphate, and aluminum hydroxide.

[0698] C138. An immunogenic composition as in C137, wherein the adjuvant is aluminum phosphate.

[0699] C139. An immunogenic composition as in C138, wherein the adjuvant is aluminum hydroxyphosphate.

[0700] C140. An immunogenic composition as in any one of C67 to C139, wherein the composition comprises a buffer, a surfactant, an excipient and an optional adjuvant, and wherein the composition is buffered to a pH of about 6.0 to about 7.0.

[0701] C141. An immunogenic composition as in any one of C67 to C140, wherein the composition comprises histidine, polysorbate - 80, NaCl and optional aluminum phosphate, and wherein the composition is buffered to a pH of about 6.0 to about 7.0.

[0702] C142. An immunogenic composition as in any one of C67 to C141, wherein the composition comprises about 10 mM to about 25 mM of histidine, about 0.01% to about 0.03% (v / w) of polysorbate - 80, about 10 mM to about 250 mM of NaCl and optional aluminum at a concentration of about 0.25 mg / ml to about 0.75 mg / ml as aluminum phosphate.

[0703] C143. An immunogenic composition as in any one of C67 to C142, wherein the composition comprises a dose of about 5 mcg / ml to about 50 mcg / ml.

[0704] C144. An immunogenic composition as in any one of C67 to C143, wherein the composition is optionally lyophilized in the presence of at least one excipient.

[0705] C145. An immunogenic composition as in C144, wherein the at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinitol, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim milk powder, glycerol, propylene glycol, water and ethanol.

[0706] C146. An immunogenic composition as in C145, wherein the at least one excipient is sucrose.

[0707] C147. An immunogenic composition as in any one of C144 to C146, wherein the composition comprises about 1% (w / v) to about 10% (w / v) of at least one excipient.

[0708] C148. An immunogenic composition according to any one of C144 to C147, wherein the composition comprises an additional excipient.

[0709] C149. An immunogenic composition according to C148, wherein the additional excipient is mannitol or glycine.

[0710] C150. An immunogenic composition according to C148 or C149, wherein the composition comprises from about 1% (w / v) to about 10% (w / v) of the additional excipient.

[0711] C151. An immunogenic composition according to any one of C143 to C150, wherein the composition is reconstituted with water, water for injection (WFI), an adjuvant suspension, or saline.

[0712] C152. An immunogenic composition according to any one of C67 to C151, for use as a medicament.

[0713] C153. An immunogenic composition according to any one of C67 to C152, in a method for inducing an immune response against GBS in a subject.

[0714] C154. An immunogenic composition according to C153, wherein the subject is a woman planning to become pregnant or a pregnant woman.

[0715] C155. An immunogenic composition according to C154, wherein the woman is in the second half of her pregnancy.

[0716] C156. An immunogenic composition according to C155, wherein the pregnant woman is at least 20 weeks pregnant.

[0717] C157. An immunogenic composition according to C156, wherein the pregnant woman is between 27 and 36 weeks pregnant.

[0718] C158. An immunogenic composition according to C157, wherein the subject is an adult 50 years of age or older.

[0719] C159. An immunogenic composition according to C158, wherein the subject is an adult 65 years of age or older.

[0720] C160. An immunogenic composition according to C159, wherein the subject is an adult 85 years of age or older.

[0721] C161. An immunogenic composition according to any one of C153 to C160, wherein the subject is immunocompromised.

[0722] C162. An immunogenic composition as in C161, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease.

[0723] C163. An immunogenic composition as in any one of C153 to C162, wherein the group B streptococcus is Streptococcus agalactiae.

[0724] C164. A method of inducing an immune response against group B streptococcus, which comprises administering to a subject an effective amount of an immunogenic composition as in any one of C67 to C150.

[0725] C165. A method of preventing or reducing a disease or condition associated with group B streptococcus in a subject, which comprises administering to the subject an effective amount of an immunogenic composition as in any one of C67 to C151.

[0726] C166. A method as in C164 or C165, wherein the subject is a female planning to become pregnant or a pregnant female.

[0727] C167. A method as in C166, wherein the female is in the second half of her pregnancy.

[0728] C168. A method as in C166 or C167, wherein the pregnant female is at least 20 weeks of gestation.

[0729] C169. A method as in any one of C166 to C168, wherein the pregnant female is at 27 to 36 weeks of gestation.

[0730] C170. A method as in C164 or C165, wherein the subject is an adult 50 years of age or older.

[0731] C171. A method as in C170, wherein the subject is an adult 65 years of age or older.

[0732] C172. A method as in C170 or C171, wherein the subject is an adult 85 years of age or older.

[0733] C173. A method as in any one of C164 to C172, wherein the subject is immunocompromised.

[0734] C174. A method as in C173, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease.

[0735] C175. A method as in any one of C164 to C174, wherein the group B streptococcus is Streptococcus agalactiae.

[0736] C176. An antibody that binds to the capsular polysaccharide in an immunogenic conjugate as in any one of C1 to C49 or C66.

[0737] C177. A composition comprising the antibody as in C176.

[0738] C178. A method for producing an antibody, which comprises administering to a subject an immunogenic composition as in any one of C67 to C151.

[0739] C179. An antibody produced by the method as in C178.

[0740] C180. A method for conferring passive immunity to a subject, which comprises the following steps:

[0741] (a) Using an immunogenic composition as in any one of C67 to C151 to produce an antibody preparation; and

[0742] (b) Administering the antibody preparation to the subject to confer passive immunity.

[0743] C181. A method for producing an immunogenic polysaccharide-protein conjugate as in any one of C1 to C49 or C66, which comprises the following steps:

[0744] (a) Reacting a GBS capsular polysaccharide with an oxidizing agent to produce an activated polysaccharide;

[0745] (b) Reacting the activated polysaccharide with a carrier protein to produce a polysaccharide-protein conjugate.

[0746] C182. The method as in C181, wherein step (b) is carried out in a polar aprotic solvent.

[0747] C183. The method as in C182, wherein the solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), sulfolane, dimethylformamide (DMF), and hexamethylphosphoramide (HMPA).

[0748] C184. The method as in C183, wherein the solvent is dimethyl sulfoxide (DMSO).

[0749] C185. The method as in any one of C181 to C184, wherein the polysaccharide reacts with 0.01 to 10.0 molar equivalents of the oxidizing agent.

[0750] C186. The method as in any one of C181 to C185, wherein the oxidizing agent is periodate.

[0751] C187. The method as in C186, wherein the periodate is sodium periodate.

[0752] C188. The method as in any one of C181 to C187, wherein the oxidation reaction in step (a) is from 1 hour to 50 hours.

[0753] C189. The method as in any one of C181 to C188, wherein the temperature of the oxidation reaction is maintained at about 2 °C to about 25 °C.

[0754] C190. The method as in any one of C181 to 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.

[0755] C191. The method as in C190, wherein the concentration of the buffer is about 1 mM to about 500 mM.

[0756] C192. The method as in any one of C181 to C191, wherein the oxidation reaction is carried out at a pH of about 4.0 to about 8.0.

[0757] C193. The method as in C181, wherein the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO).

[0758] C194. The method as in C193, wherein N-chlorosuccinimide (NCS) is a co-oxidizing agent.

[0759] C195. The method as in any one of C181 to C194, wherein step (a) further comprises quenching the oxidation reaction by adding a quenching agent.

[0760] C196. The method as in any one of C182 to C195, wherein the concentration of the polysaccharide is about 0.1 mg / mL to about 10.0 mg / mL.

[0761] C197. The method as in any one of C181 to C196, wherein the degree of oxidation of the activated polysaccharide is 5 to 25.

[0762] C198. The method as in any one of C181 to C197, wherein the method further comprises a step of lyophilizing the activated polysaccharide.

[0763] C199. The method as in 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 pinitol.

[0764] The method according to any one of C181 to C199, wherein step (b) comprises:

[0765] (1) Mixing the activated polysaccharide with a carrier protein, and

[0766] (2) Reacting the mixed activated polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate.

[0767] The method according to C200, wherein the concentration of the activated polysaccharide in step (2) is from about 0.1 mg / mL to about 10.0 mg / mL.

[0768] The method according to C200 or C201, wherein the initial ratio (weight / weight) of the activated polysaccharide to the carrier protein is from about 5:1 to 0.1:1.

[0769] The method according to any one of C200 to C202, wherein the reducing agent is selected from the group consisting of: sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride and zinc borohydride in the presence of a Bronsted acid or a Lewis acid, pyridine borane, 2-picolineborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i PrN-BH 3 benzylamine-BH 3 or 5-ethyl-2-methylpyridineborane (PEMB).

[0770] The method according to C203, wherein the reducing agent is sodium cyanoborohydride.

[0771] The method according to any one of C200 to C204, wherein the amount of the reducing agent is from about 0.1 to about 10.0 molar equivalents.

[0772] The method according to any one of C200 to C205, wherein the duration of the reduction reaction in step (2) is from 1 hour to 60 hours.

[0773] Method according to any one of C200 to C206, wherein the temperature of the reduction reaction is maintained at 10°C to 40°C.

[0774] C208. Method according to any one of C181 to C207, wherein the me...

Claims

1. An immunogenic capsular polysaccharide-carrier protein conjugate, wherein the capsular polysaccharide in the conjugate is from group B streptococcus (GBS) serotypes Ia, Ib, II, III, IV, and V, and the carrier protein is CRM197, wherein the sialic acid level of each serotype of capsular polysaccharide before conjugation is higher than 60% and the degree of oxidation is 5 - 25, and the weight ratio of capsular polysaccharide to carrier protein in each serotype of capsular polysaccharide-carrier protein conjugate is 0.5 - 3, wherein 100% sialic acid level is equivalent to 1.0 mM sialic acid per mM of capsular polysaccharide, and wherein the degree of oxidation is measured as the number of moles of sugar repeating units / moles of aldehyde.

2. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein the sialic acid level of the capsular polysaccharide before conjugation is higher than 95%.

3. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein the sialic acid level of the capsular polysaccharide before conjugation is 100%.

4. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the capsular polysaccharide has at least 0.6 mM sialic acid per mM of polysaccharide before conjugation.

5. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the capsular polysaccharide has at least 0.65 mM sialic acid per mM of polysaccharide before conjugation.

6. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the capsular polysaccharide has at least 0.7 mM sialic acid per mM of polysaccharide before conjugation.

7. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the capsular polysaccharide has at least 0.8 mM sialic acid per mM of polysaccharide before conjugation.

8. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the capsular polysaccharide has at least 0.9 mM sialic acid per mM of polysaccharide before conjugation.

9. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the molecular weight of the capsular polysaccharide is 5 kDa to 1,000 kDa.

10. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the molecular weight of the capsular polysaccharide is 25 kDa to 750 kDa.

11. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the molecular weight of the capsular polysaccharide is 25 kDa to 400 kDa.

12. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the molecular weight of the capsular polysaccharide is 100 kDa to 400 kDa.

13. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, the molecular weight of each serotype of capsular polysaccharide-carrier protein conjugate is 300 kDa to 20,000 kDa.

14. The immunogenic capsular polysaccharide-carrier protein conjugate of claim 1, wherein, The molecular weight of the capsular polysaccharide-carrier protein conjugate of each serotype is from 1,000 kDa to 15,000 kDa.

15. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 1, wherein, the molecular weight of the capsular polysaccharide-carrier protein conjugate of each serotype is from 1,000 kDa to 10,000 kDa.

16. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 1, wherein, the O-acetylation of the capsular polysaccharides from Streptococcus agalactiae serotypes Ib, III, IV, and V is from 0% to 40%, where 100% O-acetylation corresponds to 1.0 mM O-acetate per mM of sugar repeat unit.

17. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 1, wherein the O-acetylation of the capsular polysaccharide is less than 5%, where 100% O-acetylation corresponds to 1.0 mM O-acetate per mM of sugar repeat unit.

18. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 17, wherein the O-acetylation of the capsular polysaccharide is less than 3%.

19. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 17, wherein the O-acetylation of the capsular polysaccharide is less than 1%.

20. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 16, wherein, the O-acetate level of the capsular polysaccharides from Streptococcus agalactiae serotypes Ib, III, IV, and V is 0.1 mM - 0.4 mM per mM of sugar repeat unit.

21. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 20, wherein, the O-acetate level of the capsular polysaccharides from Streptococcus agalactiae serotypes Ib, III, IV, and V is 0.2 mM - 0.4 mM per mM of sugar repeat unit.

22. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 20, wherein, the O-acetate level of the capsular polysaccharides from Streptococcus agalactiae serotypes Ib, III, IV, and V is 0.3 mM - 0.4 mM per mM of sugar repeat unit.

23. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 20, wherein, the O-acetate level of the capsular polysaccharides from Streptococcus agalactiae serotypes Ib, III, IV, and V is 0.35 mM - 0.4 mM per mM of sugar repeat unit.

24. The immunogenic capsular polysaccharide-carrier protein conjugate according to claim 20, wherein, the O-acetate level of the capsular polysaccharides from Streptococcus agalactiae serotypes Ib, III, IV, and V is 0.4 mM per mM of sugar repeat unit.

25. The immunogenic capsular polysaccharide-carrier protein conjugate according to any one of claims 1 - 24, wherein the weight ratio of the capsular polysaccharide to the carrier protein in the capsular polysaccharide-carrier protein conjugate of each serotype is 0.5 - 2.

26. An immunogenic composition comprising the immunogenic capsular polysaccharide-carrier protein conjugate according to any one of claims 1 - 25.

27. The immunogenic composition of claim 26, wherein, the composition further comprises a pharmaceutically acceptable buffer, stabilizer, adjuvant, cryoprotectant, free radical oxidation inhibitor, or a mixture thereof.

28. The immunogenic composition of claim 26, wherein, the composition further comprises a salt.

29. The immunogenic composition of claim 26, wherein, the composition further comprises a carrier.

30. The immunogenic composition of claim 26, wherein, the composition further comprises a non-ionic detergent or a divalent cation.

31. The immunogenic composition of claim 26, wherein, the composition further comprises a buffer.

32. The immunogenic composition of claim 31, wherein, the buffer is selected from the group consisting of HEPES, PIPES, 2-(N-morpholino)ethanesulfonic acid (MES), Tris, phosphate, acetate, borate, citrate, glycine, histidine, and succinate.

33. The immunogenic composition of claim 31, wherein, the buffer is histidine.

34. The immunogenic composition of claim 26, wherein, the composition further comprises a surfactant.

35. The immunogenic composition of claim 34, wherein, the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters and polyoxyethylene alkyl ethers.

36. The immunogenic composition of claim 35, wherein, the surfactant is polysorbate-60, polysorbate-40, or polysorbate-20.

37. The immunogenic composition of claim 35, wherein, the surfactant is polysorbate-80.

38. The immunogenic composition of claim 26, wherein, the composition further comprises an excipient.

39. The immunogenic composition of claim 38, wherein, the excipient is selected from the group consisting of glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinitol, gelatin, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), glycerol, propylene glycol, water, and ethanol.

40. The immunogenic composition of claim 38, wherein the excipient is starch.

41. The immunogenic composition of claim 39, wherein, the excipient is sodium chloride.

42. The immunogenic composition of claim 26, wherein, the composition further comprises an adjuvant.

43. The immunogenic composition of claim 42, wherein, the adjuvant is an aluminum-based adjuvant or QS-21.

44. The immunogenic composition of claim 43, wherein, the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, hydroxyaluminum phosphate, and aluminum hydroxide.

45. The immunogenic composition of claim 44, wherein, the adjuvant is aluminum phosphate.

46. The immunogenic composition of claim 43, wherein, the adjuvant is hydroxyaluminum phosphate.

47. The immunogenic composition of claim 26, wherein, The composition comprises a buffer, a surfactant and an optional adjuvant, wherein the composition is buffered to a pH of 6.0 to 7.

0.

48. The immunogenic composition of claim 26, wherein, the composition comprises histidine, polysorbate-80, sodium chloride and optionally aluminum phosphate, wherein the composition is buffered to a pH of 6.0 to 7.

0.

49. The immunogenic composition of claim 26, wherein, the composition comprises 10 mM to 25 mM of histidine, 0.01% to 0.03% (v / w) of polysorbate-80, 10 mM to 250 mM of sodium chloride and optionally 0.25 mg / ml to 0.75 mg / ml of aluminum phosphate.

50. The immunogenic composition of claim 26, wherein, the composition comprises a dose of 5 mcg / ml to 50 mcg / ml.

51. The immunogenic composition of claim 26, wherein, the composition is lyophilized, optionally in the presence of at least one excipient.

52. The immunogenic composition of claim 51, wherein, the at least one excipient is selected from the group consisting of glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinitol, gelatin, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), glycerol, propylene glycol, water and ethanol.

53. The immunogenic composition of claim 51, wherein, the at least one excipient is starch.

54. The immunogenic composition of claim 52, wherein, the at least one excipient is sucrose.

55. The immunogenic composition of claim 51, wherein, the composition comprises 1% (w / v) to 10% (w / v) of at least one excipient.

56. The immunogenic composition of claim 51, wherein, the composition comprises an additional excipient.

57. The immunogenic composition of claim 56, wherein, the additional excipient is mannitol or glycine.

58. The immunogenic composition of claim 56, wherein, the composition comprises 1% (w / v) to 10% (w / v) of the additional excipient.

59. The immunogenic composition of any one of claims 26 - 58, wherein, the composition is reconstituted with water, an adjuvant suspension or saline.

60. The immunogenic composition of any one of claims 26 - 58, wherein, the composition is reconstituted with water for injection.

61. Use of the immunogenic composition of any one of claims 26 - 60 in the manufacture of a medicament for conferring passive immunity to a subject, wherein the medicament induces an immune response against group B streptococcus in the subject.

62. A method of making a capsular polysaccharide - carrier protein conjugate as defined in any one of claims 1 - 25, comprising the steps of: (a) reacting a group B streptococcus capsular polysaccharide with an oxidizing agent to produce an activated capsular polysaccharide; and (b) reacting the activated capsular polysaccharide with a carrier protein to produce a capsular polysaccharide - carrier protein conjugate.

63. The method of claim 62, which comprises the following steps: (a) Reacting the isolated group B streptococcus capsular polysaccharide with an oxidizing agent; (b) Quenching the oxidation reaction of step (a) by adding a quenching agent to produce the activated group B streptococcus capsular polysaccharide; (c) Mixing the activated group B streptococcus capsular polysaccharide with a carrier protein, (d) Reacting the mixed activated group B streptococcus capsular polysaccharide and carrier protein with a reducing agent to form a group B streptococcus capsular polysaccharide - carrier protein conjugate, and (e) The unreacted aldehydes were capped by adding sodium borohydride (NaBH 4 ). wherein, steps (c) and (d) are carried out in DMSO.

64. Use of an immunogenic composition according to any one of claims 26 - 60 in the preparation of a medicament for preventing or alleviating invasive group B streptococcus infection in a subject.

65. The use according to claim 64, wherein, the subject is a woman planning to become pregnant or a pregnant woman.

66. The use according to claim 65, wherein, the woman is in the second half of her pregnancy.

67. The use according to claim 65, wherein, the woman is at least 20 weeks pregnant.

68. The use according to claim 65, wherein, the woman is between 27 and 36 weeks pregnant.

69. The use according to claim 64, wherein, the subject is an adult 50 years old or older.

70. The use according to claim 64, wherein, the subject is an adult 65 years old or older.

71. The use according to claim 64, wherein, the subject is an adult 85 years old or older.

72. The use according to any one of claims 64 - 71, wherein, the subject is immunocompromised.

73. The use according to claim 72, wherein, the subject has a medical condition selected from the group consisting of: HIV infection, cancer, cardiovascular disease, or liver disease.

74. The use according to claim 72, wherein, the subject has obesity.

75. The use according to claim 72, wherein, the subject has diabetes.

Images