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

Abstract

To provide methods and compositions for reducing or preventing invasive GBS disease in subjects.SOLUTION: The invention relates to compositions comprising immunogenic polysaccharide-protein conjugates comprising a capsular polysaccharide (CP) from Streptococcus agalactiae, commonly referred to as group B streptococcus (GBS), and a carrier protein, wherein the CP is selected from a group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX, and wherein the CP has a sialic acid level of greater than about 60%. The invention also relates to methods of making the conjugates and immunogenic compositions comprising the conjugates. The invention further relates to methods for inducing an immune response in subjects against GBS and / or for reducing or preventing invasive GBS disease in subjects using the compositions disclosed herein.SELECTED DRAWING: None

Description

[Technical field]

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

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

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

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

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

[0006] The structures of the polysaccharides that coat bacteria vary widely even within a single species of bacteria. For example, there are 10 different serotypes of GBS (i.e., serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX) due to variations in the bacterial polysaccharide capsule. It is therefore desirable for a polysaccharide-based vaccine to consist of a panel of polysaccharides to ensure broad coverage of the different circulating strains.

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

[0008] Individual monovalent polysaccharide-protein conjugates of GBS serotypes Ia, Ib, II, III, and V have been evaluated in phase I and II clinical trials in non-pregnant adults (Brigtsen, AK, et al., Journal of Infectious Diseases, 185(9):1277-1284 (2002); Baker, CJ, et al., J. Infect. Dis., 188(1):66-73 (2003); Baker, CJ, et al., J. Infect. Dis., 189(6):1103-1112 (2004); Baker, CJ, et al., Vaccine, 25(1):55-63 (2007)). Bivalent II-TT and III-TT glycoconjugate vaccines, as well as Ia-CRM 197 , Ib-CRM 197 and III-CRM 197 Trivalent vaccines containing glycoconjugates have also been investigated (Baker JID 2003; Clicaltrials.gov NCT01193920, NCT01412801 and NCT01446289). However, no GBS vaccine has yet been approved.

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

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

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

[0012] Studies of GBS epidemiology have found that there is geographic variation in serotype prevalence and indicate that the prevalence of non-GBS6 serotypes depends on a number of factors, including topography, age of the patient, and whether surveillance is based on colonization or invasive disease.

[0013] For example, serotype VI and VIII isolates were shown to be the predominant invasive species in healthy pregnant women in Japan (Lachenauer, CS, et al., JID 179(4):1030-1033 (1999)). The rate of non-GBS6 serotype colonization is significantly higher in Asia than in Western countries. In a Japanese study of 73 pregnant women, the rates of serotype VI and VIII carriage were 35.6% and 24.7%, respectively. In contrast, these serotypes are rarely observed among pregnant women in the United States [1, 2]. However, these serotypes do not cause correspondingly high disease rates in infants. A large-scale study of GBS epidemiology in Japan in which isolates causing neonatal disease were collected between 2011 and 2015 (n= In 132 cases, the prevalence of non-GBS6 serotypes was negligible (1.1% for VI in EOD only; 1.1% for serotype IX in late-onset disease only) [3]. These results are in line with a study of early neonatal invasive isolates (n=60), which found similar serotype prevalence: III (48.3%), Ia (30.0%), and Ib (10.0%) [4]. This trend for a low prevalence of non-GBS6 neonatal disease appears to be similar to that in China, although studies published to date have involved smaller numbers of isolates (<50) [5-7].

[0014] Significantly, the rate of invasive disease caused by non-GBS6 serotypes is much higher in elderly patients. In a large Japanese study (n=443) based on isolates obtained between 2010 and 2013, the prevalence of invasive serotype VI was 9.5% [8]. A similar trend was observed in Taiwan, where a high rate of invasive serotype VI disease was seen in elderly people but not infants [9].

[0015] Based on these surveillance studies, serotype VI appears to be an emerging threat that would prompt its inclusion in second-generation vaccines, especially those targeted at older populations. The incidence of invasive disease caused by serotypes VII, VIII, and IX is currently rare but could become more significant if serotype replacement occurs following the introduction of the GBS6 vaccine. Summary of the Invention [Problem to be solved by the invention]

[0016] Thus, there is a need for polysaccharide-protein conjugate vaccines or monoclonal antibodies to confer passive immunity as a means to prevent or treat GBS disease, including that caused by the newly emerged serotypes VI, VII, VIII and IX, among broad populations worldwide. [Means for solving the problem]

[0017] In one embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising Group B Streptococcus (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%. In another embodiment, the invention comprises an immunogenic composition as described herein, wherein the capsular polysaccharide is selected from the group consisting of serotypes VI, VII, VIII and IX.

[0018] In yet another embodiment, the invention includes an immunogenic composition as described herein, wherein the capsular polysaccharide has at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 mM sialic acid per mM polysaccharide.

[0019] In yet another embodiment, the invention includes an immunogenic composition as described herein, wherein the capsular polysaccharide has a molecular weight of between about 5 kDa and about 1,000 kDa, between about 25 kDa and about 750 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 200 kDa, or between about 100 kDa and about 400 kDa.

[0020] In another embodiment, the invention includes an immunogenic composition as described herein, wherein the molecular weight of the conjugate is between about 300 kDa and about 20,000 kDa, between about 1,000 kDa and about 15,000 kDa, or between about 1,000 kDa and about 10,000 kDa.

[0021] In further embodiments, the invention includes an immunogenic composition as described, wherein the capsular polysaccharides are less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% O-acetylated.

[0022] In another embodiment, the invention comprises an immunogenic composition as described, wherein the capsular polysaccharide has at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.35 or about 0.4 mM O-acetate per mM saccharide repeat unit.

[0023] In one embodiment, the present invention relates to a method for producing a carrier protein comprising the steps of: 197 , diphtheria toxoid (DT), tetanus toxoid (TT) and streptococcal C5a peptidase (SCP), as described herein.

[0024] In another embodiment, the invention includes a method of isolating capsular polysaccharide comprising reacting an organic reagent with a cell broth containing capsular polysaccharide producing bacteria.

[0025] In another embodiment, the invention includes a method for isolating capsular polysaccharides as described that does not lyse the bacteria and / or heat kill the bacteria.

[0026] In another embodiment, the invention includes a method of isolating capsular polysaccharides as described, further comprising the step of centrifugation to provide a cell paste.

[0027] In yet another embodiment, the invention includes a method of isolating capsular polysaccharides as described further comprising a filtering step, which may include a filtering step that is hemodiafiltration.

[0028] In yet another embodiment, the invention includes a method of isolating capsular polysaccharide as described, wherein the capsular polysaccharide producing bacteria comprises Streptococcus agalactiae.

[0029] In another embodiment, the invention includes a method of isolating capsular polysaccharides as described, wherein the pH of the reaction is from about 5.5 to about 9.5.

[0030] In yet another embodiment, the invention includes a method of isolating capsular polysaccharides as described, wherein the reaction occurs at a temperature of from about 20°C to about 85°C.

[0031] In yet another embodiment, the invention includes a method for isolating capsular polysaccharides as described, wherein the reaction time is from about 10 hours to about 90 hours.

[0032] In another embodiment, the invention includes a method of making an immunogenic composition as described herein, wherein the capsular polysaccharide is isolated according to a method as described herein.

[0033] In certain embodiments, the present invention comprises an immunogenic composition comprising a capsular polysaccharide-protein conjugate prepared by the methods as described herein.

[0034] In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide from Group B Streptococcus (GBS) serotype VI and a carrier protein, and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, IV, V, VII, VIII and IX.

[0035] In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VI and a carrier protein, In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VI, further comprising at least one additional serotype selected from Ia, Ib, II, III, IV, V, VII, VIII and IX.

[0036] In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS serotype VI capsular polysaccharide, further comprising at least one additional serotype selected from Ia, Ib, II, III, IV and V. In a further embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS serotype VI capsular polysaccharide, further comprising at least one additional serotype selected from VII, VIII and IX.

[0037] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises a capsular polysaccharide from Group B Streptococcus (GBS) serotype VI and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%.

[0038] In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VII and a carrier protein, In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VII, further comprising at least one additional serotype selected from Ia, Ib, II, III, IV, V, VI, VIII and IX.

[0039] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises a capsular polysaccharide derived from Group B Streptococcus (GBS) serotype VII capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%.

[0040] In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VIII and a carrier protein, In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VIII, further comprising at least one additional serotype selected from Ia, Ib, II, III, IV, V, VI, VII and IX.

[0041] In another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises a capsular polysaccharide derived from Group B Streptococcus (GBS) serotype VIII capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%.

[0042] In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype IX and a carrier protein, In another embodiment, the invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype IX, further comprising at least one additional serotype selected from Ia, Ib, II, III, IV, V, VI, VII and VIII.

[0043] In a further embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises a capsular polysaccharide derived from Group B Streptococcus (GBS) serotype IX capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%.

[0044] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VI and VII.

[0045] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VI and VIII.

[0046] In another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VI and IX.

[0047] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VII and VIII.

[0048] In one embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VII and IX.

[0049] In another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VIII and IX.

[0050] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VI, VII and VIII.

[0051] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VI, VII and IX.

[0052] In one embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VI, VIII and IX.

[0053] In another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes VI, VII, VIII and IX.

[0054] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes Ia, Ib, II, III, IV, V and VI.

[0055] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes Ia, Ib, II, III, IV, V, VI and VII.

[0056] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes Ia, Ib, II, III, IV, V, VI, VII and VIII.

[0057] In one embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

[0058] In one embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharide from GBS serotypes Ia and VI and a carrier protein.

[0059] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes Ib and VI.

[0060] In yet another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes II and VI.

[0061] In another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharides from GBS serotypes III and VI and a carrier protein.

[0062] In a further embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharide and a carrier protein from GBS serotypes IV and VI.

[0063] In another embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises capsular polysaccharides from GBS serotypes V and VI and a carrier protein.

[0064] In another embodiment, the invention includes an immunogenic composition as described herein, further comprising a pharma- ceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic surfactant, inhibitor of free radical oxidation, carrier, or mixtures thereof.

[0065] In yet another embodiment, the invention includes an immunogenic composition as described herein, further comprising a buffer selected from the group consisting of HEPES, PIPES, MES, Tris(trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate.

[0066] In yet another embodiment, the invention comprises an immunogenic composition as described herein, further comprising a surfactant selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20 and polyoxyethylene alkyl ethers.

[0067] In certain embodiments, the invention includes an immunogenic composition as described herein, further comprising an excipient selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone flour, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol.

[0068] In another embodiment, the invention comprises an immunogenic composition as described herein, further comprising an adjuvant selected from Streptococcal C5a peptidase (SCP), an aluminum-based adjuvant or QS-21, wherein the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyl phosphate and aluminum hydroxide.

[0069] In one embodiment, the invention includes an immunogenic composition as described herein, comprising a buffer, a surfactant, an excipient, and optionally an adjuvant, and buffered to a pH of about 6.0 to about 7.0.

[0070] In yet another embodiment, the invention includes an immunogenic composition as described herein comprising histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, buffered to a pH of about 6.0 to about 7.0.

[0071] In yet another embodiment, the invention comprises an immunogenic composition as described herein comprising about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate.

[0072] In a further embodiment, the invention comprises an immunogenic composition as described herein comprising a dose of about 5 mcg / ml to about 50 mcg / ml.

[0073] In one embodiment, the invention comprises an immunogenic composition as described herein, optionally lyophilized in the presence of at least one excipient, wherein the at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone flour, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol.

[0074] In yet another embodiment, the invention comprises an immunogenic composition as described herein comprising about 1% (w / v) to about 10% (w / v) of at least one excipient.

[0075] In another embodiment, the invention includes an immunogenic composition as described herein, further comprising an additional excipient selected from mannitol or glycine, comprising about 1% (w / v) to about 10% (w / v) of the additional excipient.

[0076] In certain embodiments, the invention comprises an immunogenic composition as described herein that is reconstituted with water, water for injection (WFI), an adjuvant suspension, or saline.

[0077] In another embodiment, the present invention comprises an immunogenic composition as described herein for use as a medicament.

[0078] In yet another embodiment, the invention comprises an immunogenic composition as described herein for use in a method of eliciting an immune response against GBS in a subject.

[0079] In yet another embodiment, the invention includes an immunogenic composition as described herein, wherein the subject is a woman planning a pregnancy or a pregnant woman, optionally in the second half of pregnancy, at least 20 weeks pregnant, or between 27 and 36 weeks pregnant.

[0080] In another embodiment, the invention includes an immunogenic composition as described herein, wherein the subject is an adult aged 50 or more, 65 or more, or 85 or more.

[0081] In certain embodiments, the invention includes an immunogenic composition as described herein, wherein the subject is immunocompromised and / or the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

[0082] In yet another embodiment, the invention comprises an immunogenic composition as described herein, wherein the Group B Streptococcus is Streptococcus agalactiae.

[0083] In yet another embodiment, the invention includes a method of inducing an immune response against Group B Streptococcus, comprising administering to a subject an effective amount of an immunogenic composition as described herein.

[0084] In another embodiment, the present invention includes a method of preventing or reducing a disease or condition associated with Group B Streptococcus in a subject, comprising the step of administering to the subject an effective amount of an immunogenic composition as described herein.

[0085] In yet another embodiment, the invention includes a method of preventing or reducing a disease or condition associated with Group B Streptococcus in a subject, comprising the step of administering to the subject an effective amount of an immunogenic composition as described herein, wherein the subject is a woman planning a pregnancy or a pregnant woman, optionally wherein the woman is in the second half of pregnancy, at least 20 weeks pregnant, or between 27 and 36 weeks pregnant.

[0086] In another embodiment, the invention includes a method of preventing or reducing a disease or condition associated with Group B Streptococcus in a subject, comprising the step of administering to the subject an effective amount of an immunogenic composition as described herein, wherein the subject is an adult aged 50 years or older, 65 years or older, or 85 years or older, and / or the subject is immunocompromised, and optionally the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

[0087] In certain embodiments, the present invention includes a method of preventing or reducing a disease or condition associated with Group B Streptococcus in a subject, comprising the step of administering to the subject an effective amount of an immunogenic composition as described herein, wherein the Group B Streptococcus is Streptococcus agalactiae.

[0088] In another embodiment, the invention includes a method of eliciting an immune response against Group B Streptococcus serotype V or serotype VI or serotype VII or serotype VIII or serotype IX, comprising the step of administering to a subject an immunogenic composition as described herein.

[0089] In yet another embodiment, the invention comprises an antibody that binds to a capsular polysaccharide in an immunogenic conjugate as described herein.

[0090] In a further embodiment, the invention includes a composition comprising an antibody as described herein, or a method of producing an antibody, comprising administering to a subject an immunogenic composition as described herein.

[0091] In certain embodiments, the invention includes a method of conferring passive immunity to a subject, the method comprising producing an antibody preparation using an immunogenic composition as described herein, and administering the antibody preparation to the subject to confer passive immunity.

[0092] In another embodiment, the present invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, comprising the steps of (a) reacting a GBS capsular polysaccharide with an oxidizing agent to provide an activated polysaccharide, and (b) reacting the activated polysaccharide with a carrier protein to provide a polysaccharide-protein conjugate, wherein step (b) may be carried out in a polar aprotic solvent selected from the group consisting of dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF) and hexamethylphosphoramide (HMPA).

[0093] In one embodiment, the invention includes a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein the polysaccharide is reacted with 0.01 to 10.0 molar equivalents of an oxidizing agent, wherein the oxidizing agent is a periodate, which may include sodium periodate.

[0094] In another embodiment, the present invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein the oxidation reaction of step (a) is for between 1 hour and 50 hours, the temperature of the oxidation reaction may be maintained between about 2° C. and about 25° C., the oxidation reaction may be carried out in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) and Bis-Tris, and further, the buffer may have a concentration between about 1 mM and about 500 mM.

[0095] In yet another embodiment, the invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein the oxidation reaction is carried out at a pH between about 4.0 and about 8.0, the oxidizing agent may be 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), N-chlorosuccinimide (NCS) may be a co-oxidizing agent, and / or step (a) further comprises the step of further quenching the oxidation reaction by addition of a quenching agent.

[0096] In another embodiment, the present invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein the concentration of the polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL and the degree of oxidation of the activated polysaccharide may be between 5 and 25.

[0097] In yet another embodiment, the present invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, further comprising the step of lyophilizing the activated polysaccharide in the presence of a sugar selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit.

[0098] In a further embodiment, the present invention includes a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein step (b) comprises combining an activated polysaccharide with a carrier protein and reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate, wherein the concentration of the activated polysaccharide in step (b) may be between about 0.1 mg / mL and about 10.0 mg / mL and / or the initial ratio of activated polysaccharide to carrier protein (wt / wt) may be between 5:1 and 0.1:1.

[0099] In yet a further embodiment, the present invention relates to a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein the reducing agent is a Bronsted or Lewis acid, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i The method includes the step of reducing the aryl group of the aryl group by reacting the aryl group with PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB) in the presence of PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB) in an amount of between about 0.1 and about 10.0 molar equivalents.

[0100] In certain embodiments, the present invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein the duration of the reduction reaction in step (2) is between 1 hour and 60 hours and / or the temperature of the reduction reaction is maintained between 10° C. and 40° C.

[0101] In a further embodiment, the present invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, further comprising a step of capping unreacted aldehydes by addition of borohydride (step (c)), wherein the amount of borohydride may be between about 0.1 and about 10.0 molar equivalents, wherein the borohydride is selected from the group consisting of sodium borohydride (NaBH4), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride and magnesium borohydride, wherein the duration of the capping step may be between 0.1 hours and 10 hours, and / or wherein the temperature of the capping step is maintained between about 15° C. and about 45° C.

[0102] In another embodiment, the present invention comprises a method of making an immunogenic polysaccharide-protein conjugate as described herein, wherein the polysaccharide-protein conjugate comprises less than about 40% free polysaccharide compared to the total amount of polysaccharide.

[0103] In yet another embodiment, the present invention comprises a method of making a polysaccharide-protein conjugate as described herein, wherein the ratio of polysaccharide to carrier protein (w / w) in the conjugate is between about 0.5 and about 3.0 and / or the degree of conjugation of the conjugate is between 2 and 15.

[0104] In yet another embodiment, the invention comprises a method of making a polysaccharide-protein conjugate as described herein comprising the steps of: (a) reacting isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction of step (a) by the addition of a quenching agent to provide activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; and (e) capping any unreacted aldehydes by the addition of sodium borohydride (NaBH4), wherein steps (c) and (d) are performed in DMSO.

[0105] In certain embodiments, the invention includes a method of making a polysaccharide-protein conjugate as described herein, comprising the steps of: (a) reacting isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction of step (a) by the addition of a quenching agent to result in activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; (e) capping unreacted aldehydes by the addition of sodium borohydride (NaBH4); and (f) purifying the polysaccharide-protein conjugate, wherein steps (c) and (d) are performed in DMSO. [Brief description of the drawings]

[0106] [Figure 1] FIG. 1 shows immunogenicity and cross-reactivity of GBS capsular polysaccharide serotype VI conjugates. [Figure 2A] FIG. 1 shows the immunogenicity of GBS capsular polysaccharide serotype VII conjugates. [Figure 2B] FIG. 1 shows the immunogenicity of GBS capsular polysaccharide serotype VIII conjugates. [Figure 2C] FIG. 1 shows the immunogenicity of GBS capsular polysaccharide serotype IX conjugates. [Figure 3A] FIG. 1 shows immunogenicity and cross-reactivity of GBS capsular polysaccharide serotype VII conjugates. [Figure 3B] FIG. 1 shows immunogenicity and cross-reactivity of GBS capsular polysaccharide serotype IX conjugates. [Figure 4] FIG. 1 shows immunogenicity of multivalent GBS conjugate vaccines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] This invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0108] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

[0109] The terms used herein have meanings that are recognized and known to those of ordinary skill in the art; however, for convenience and completeness, certain terms and their meanings are explained below and throughout the specification.

[0110] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps described herein and / or that would become apparent to those skilled in the art upon reading this disclosure and so forth.

[0111] The term "about" or "approximately" means within a statistically meaningful range of values. Such a range may be within an order of magnitude, typically within 20%, and more typically within 10%, and even more typically within 5% of a given value or range. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily appreciated by one of ordinary skill in the art. Whenever a range is described within this application, all integers within the range are also contemplated as embodiments of the invention.

[0112] It should be noted that in this disclosure, terms such as "comprises", "includes", "including", "containing", "containing" and the like may have the meanings ascribed to them in the United States Patent Law, e.g., "includes", "included", "comprising", etc. Such terms refer to the inclusion of a particular ingredient or set of ingredients without excluding any other elements. Terms such as "consisting essentially of" and "consisting essentially of" have the meanings ascribed to them in the United States Patent Law, e.g., allowing for the inclusion of additional ingredients or steps that do not depart from the novel or basic features of the invention, i.e., excluding additional unrecited ingredients or steps that depart from the novel or basic features of the invention, and excluding prior art ingredients or steps, such as documents in the art cited or incorporated by reference herein, particularly where the goal of this document is to define an embodiment that is patentable, e.g., novel, non-obvious, inventive, goes beyond the prior art, e.g., documents cited or incorporated by reference herein. Also, the terms "consisting of" and "consisting of" have the meanings ascribed to them in the United States Patent Law, i.e., these terms are closed-ended. Thus, these terms refer to the inclusion of a particular ingredient or set of ingredients and the exclusion of all others.

[0113] The term "antigen" generally refers to an immunogenic substance capable of stimulating the production of an antibody or a T-cell response or both in an animal, including a biological molecule, usually a protein, peptide, polysaccharide, lipid or conjugate, that contains at least one epitope to which a cognate antibody can selectively bind, or in some cases a composition that is injected or absorbed into an animal. The immune response may be generated against the whole molecule or against one or more various portions of the molecule (e.g., epitopes or haptens). The term may be used to refer to individual molecules or homogeneous or heterogeneous populations of antigenic molecules. Antigens are recognized by antibodies, T-cell receptors, or other elements of specific humoral and / or cellular immunity. The term "antigen" encompasses all related antigenic epitopes. Epitopes of a given antigen can be identified using any number of epitope mapping techniques well known to those of skill in the art (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, ed., 1996) Humana Press, Totowa, NJ). For example, linear epitopes can be determined, e.g., by simultaneously synthesizing a large number of peptides on a solid support, peptides corresponding to portions of a protein molecule, and reacting the peptides with an antibody while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen, HM et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1984); Geysen, HM et al., Molec. Immunol., 23(7):709-715 (1986), all of which are incorporated herein by reference in their entireties. Similarly, conformational epitopes can be identified by determining the spatial conformation of amino acids, such as by, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols, supra).Furthermore, for purposes of the present invention, "antigen" may also be used to refer to proteins that include modifications (generally conservative in nature, but which may be non-conservative) to the native sequence, such as deletions, additions, and substitutions, so long as the protein maintains its ability to elicit an immunological response. These modifications may be deliberate, such as through site-directed mutagenesis, or through specific synthetic procedures, or through genetic engineering approaches, or may be accidental, such as through mutations of the host that produces the antigen. Furthermore, antigens may be derived, obtained, or isolated from microorganisms, such as bacteria, or may be whole organisms. Similarly, oligonucleotides or polynucleotides expressing antigens, such as in nucleic acid immunization applications, are also included in the definition. Synthetic antigens, such as polyepitopes, contiguous epitopes, and other recombinant or synthetically derived antigens, are also encompassed (Bergmann, C. et al., Eur. J. Immunol., 23(11):2777-2781 (1993); Bergmann, CC et al., J. Immunol., 157(8):3242-3249 (1996); Suhrbier, A., Immunol. and Cell Biol., 75(4):402-408 (1997)).

[0114] The terms "vaccine" or "vaccine composition" are used interchangeably and refer to a pharmaceutical composition that includes at least one immunogenic composition that elicits an immune response in an animal.

[0115] capsular polysaccharide As used herein, the term "sugar" refers to a single sugar moiety or unit, as well as combinations of two or more single sugar moieties or units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. The term "sugar" may be used interchangeably with the term "carbohydrate." Polysaccharides may be linear or branched.

[0116] "Monosaccharide" as used herein refers to a single sugar residue in an oligosaccharide. The term "disaccharide" as used herein refers to a polysaccharide composed of two monosaccharide units or moieties linked together by a glycosidic bond.

[0117] In one embodiment, the polysaccharide is an oligosaccharide (OS). "Oligosaccharide" as used herein refers to a compound containing two or more monosaccharide units or moieties. Within the context of an oligosaccharide, each monomer unit or moiety is a monosaccharide that is or can be linked to another monosaccharide unit or moiety via a hydroxyl group. Oligosaccharides can be prepared either by chemical synthesis from protected single residue sugars or by chemical degradation of biologically produced polysaccharides. Alternatively, oligosaccharides can be prepared by in vitro enzymatic methods.

[0118] In a preferred embodiment, the polysaccharide is a polysaccharide (PS), which refers to a linear or branched polymer of at least five monosaccharide units or moieties. For clarity, larger numbers of repeating units (where n is greater than about 5, such as greater than about 10) are referred to herein as polysaccharides.

[0119] In one embodiment, the polysaccharide is a cell surface polysaccharide. Cell surface polysaccharide refers to a polysaccharide having at least a portion located on the outermost bacterial cell membrane or bacterial cell surface, including the peptidoglycan layer, cell wall and capsule. Typically, cell surface polysaccharides are associated with eliciting an immune response in vivo. Cell surface polysaccharides may be "cell wall polysaccharides" or "capsular polysaccharides". Cell wall polysaccharides typically form a discontinuous layer on the bacterial surface.

[0120] In one embodiment, the polysaccharide is a capsular polysaccharide. Capsular polysaccharides are glycopolymers that include repeating units of one or more monosaccharides joined by glycosidic bonds. Capsular polysaccharides typically form a capsule-like layer around the bacterial cell. "Capsular polysaccharide" or "capsule polysaccharide" refers to the polysaccharide capsule that is on the exterior of the cell wall of most isolates of streptococci. For example, all GBS capsular polysaccharides have a branched repeating structure with terminal α2-3-linked sialic acid that is required for bacterial virulence. Capsular-associated sialic acid (quantified by HPLC assay) was detected in more than 94% of invasive neonatal isolates from TEST cultured in vitro.

[0121] The present inventors have found that the sialic acid level of GBS capsular polysaccharide is an important feature for generating an immune response. Prior disclosures have provided conflicting information regarding the sialic acid level for serotype V, only the observation that desialylated serotype V is preferred (International Patent Application Publication No. WO2012 / 035519), and that a sialic acid content of more than 50% for serotype V can be used (International Patent Application Publication No. WO2014 / 053612). However, none of these references describe the importance of the sialic acid level for at least the majority of the GBS polysaccharide to immunogenicity. The present inventors have surprisingly found that GBS capsular polysaccharide requires about 60% or more sialic acid prior to conjugation to provide an immune response comparable to that of a polysaccharide with a natural sialic acid level (i.e., 100% or more than about 95%). Even a sialic acid level of 58%, which is within the previously disclosed range for serotype V, adversely affected immunogenicity.

[0122] Thus, in one embodiment of the invention, the capsular polysaccharides comprise their native sialic acid levels of about 100% or greater than about 95%, etc. In another embodiment, the capsular polysaccharides may be desialylated, such as up to about 40% (greater than about 60% sialylation level), for example up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), and up to about 5% (greater than about 95% sialylation level).

[0123] It should be noted that a sialic acid level of 100% corresponds to about 1.0 mM sialic acid per mM polysaccharide. Thus, a capsular polysaccharide may have about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide. In further embodiments, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, e.g., at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0124] The terminal sialic residues of some capsular polysaccharide (CP) serotypes are partially O-acetylated (OAc) (Lewis, AL et al., Proceedings of the National Academy of Sciences USA, 101(30):11123-8 (2004)). Serotypes Ib, III, IV, V, VI and IX are partially O-acetylated (up to about 40%), whereas serotypes Ia, II and VII have little or no O-acetylation (less than about 5%) (Lewis 2004). In one embodiment of the invention, the capsular polysaccharides comprise their natural O-acetylation level (from about 0% to about 40%). In another embodiment, the capsular polysaccharides may be O-deacetylated (less than about 5%). The degree of O-acetylation of polysaccharides or oligosaccharides can be determined by any method known in the art, for example, by proton NMR (Lemercinier, X. et al., Carbohydrate Research, 296:83-96 (1996); Jones, C. et al., Journal of Pharmaceutical and Biomedical Analysis, 30:1233-1247 (2002); International Patent Application Publication Nos. WO2005 / 033148 and WO00 / 56357). Another commonly used method is described by Hestrin, S., J. Biol. Chem., 180:249-261 (1949).

[0125] Additionally, 100% O-acetate corresponds to about 1.0 mM O-acetate per mM saccharide repeat unit. Thus, a partially O-acetylated polysaccharide contains at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM O-acetate per mM saccharide repeat unit. A deacetylated O-polysaccharide contains less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM saccharide repeat unit.

[0126] Streptococcal microorganisms capable of causing invasive disease are also generally capable of producing CPs that encapsulate the bacteria and enhance its resistance to clearance by the host's innate immune system. The CPs serve to encase the bacterial cells in a protective capsule that renders the bacteria resistant to phagocytosis and intracellular killing. Bacteria lacking a capsule are more susceptible to phagocytosis. Capsular polysaccharides are frequently important virulence factors for many bacterial pathogens, including Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Staphylococcus aureus.

[0127] Capsular polysaccharides can be used to serotype specific species of bacteria. Typing to specific structures or unique epitope features of the capsular polysaccharide is usually accomplished by reaction with specific antisera or monoclonal antibodies produced. There are 10 GBS serotypes: Ia, Ib, and II-IX (Ferrieri, P. et al., Emerg. Infect. Dis. [Internet], 19(4) (2013), available at http: / / wwwnc.cdc.gov / eid / article / 19 / 4 / 12-1572_article).

[0128] In one embodiment of the invention, the polysaccharide is isolated from Streptococcus agalactiae. The polysaccharide may be isolated from any encapsulated strain of S. agalactiae, such as 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524, MB4052 (ATCC Accession No. 31574), H36B (ATCC Accession No. 12401), S40, S42, MB4053 (ATCC Accession No. 31575), M709, 133, 709, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1111, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1310, 1320, 1330, 1400, 1410 357, PFEGBST0267, MB4055 (ATCC Accession No. 31576), 18RS21 (ATCC Accession No. BAA-1175), S16, S20, V8 (ATCC Accession No. 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB4082 (ATCC Accession No. 31577), M132, 110, M781 (ATCC Accession No. BAA-22), D136C(3)(ATCC Accession No. C accession no. 12403), M782, S23, 120, MB4316 (M-732; ATCC accession no. 31475), M132, K79, COH1 (ATCC accession no. BAA-1176), PFEGBST0563, 3139 (ATCC accession no. 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession no. BAA-23), CJB112, 2603V / R (ATCC Accession No. BAA-611), NCTC10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B4589, B4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62 and IT-PW-64, etc.

[0129] The polysaccharides described herein may be isolated by methods known in the art, including, for example, the methods described herein. As used herein, "isolated" refers to being obtained and separated from a particular source. The term "isolated" further refers to being free from its respective naturally occurring form, situation and / or environment. For example, "isolated from streptococci" refers to a material obtained and separated from streptococcal cells. An isolated polysaccharide is not naturally occurring. The term "isolated" means that the material has been removed from its original environment (e.g., the natural environment if it is naturally occurring, or from its host organism if it is a recombinant entity, or transported from one environment to a different environment). For example, an "isolated" capsular polysaccharide, protein or peptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or is substantially free of chemical precursors or other chemicals if chemically synthesized or present in a mixture as part of a chemical reaction. In the present invention, the protein or polysaccharide may be isolated from bacterial cells or from cell debris so as to be provided in a form useful in the manufacture of an immunogenic composition. The term "isolated" or "isolating" may include purifying or purification, including methods for purifying isolated polysaccharides known in the art and / or described herein. The term "substantially free of cellular material" includes preparations of a polypeptide / protein in which the polypeptide / protein is separated from cellular components of the cells from which it was isolated or recombinantly produced. Thus, a capsular polysaccharide, protein or peptide that is substantially free of cellular material includes preparations of capsular polysaccharide, protein or peptide having less than about 30%, 20%, 10%, 5%, 2.5% or 1% (by dry weight) of contaminating proteins or polysaccharides or other cellular material. If the polypeptide / protein is recombinantly produced, it is preferably also substantially free of culture medium, i.e., culture medium represents less than about 20%, 10% or 5% of the volume of the protein preparation.When a polypeptide / protein or polysaccharide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein or polysaccharide. Thus, such preparations of a polypeptide / protein or polysaccharide have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide / protein or polysaccharide fragment of interest.

[0130] In one embodiment of the invention, the polysaccharide is isolated from bacteria. In another embodiment of the invention, the polysaccharide is recombinantly produced. In a further embodiment, the polysaccharide is synthetic or chemically synthesized according to conventional methods. In yet another embodiment of the invention, the polysaccharide is prepared by cloning and expressing a biosynthetic pathway for producing the polysaccharide followed by expression in a surrogate host. In one embodiment, the polysaccharide is immunogenic. For example, the inventors have discovered that each of the polysaccharides described herein is capable of eliciting or eliciting an immune response. The term "immunogenic" refers to the ability to initiate, trigger, cause, enhance, improve, and / or enhance a humoral and / or cell-mediated immune response in a mammal. In one embodiment, the mammal is a human, a primate, a rabbit, a pig, a mouse, etc.

[0131] The molecular weight of the capsular polysaccharide is a consideration for use in immunogenic compositions. High molecular weight capsular polysaccharides can elicit certain antibody immune responses due to the higher valency epitopes present on the antigen surface. Isolation and purification of high molecular weight capsular polysaccharides are contemplated for use in the conjugates, compositions and methods of the invention.

[0132] However, in one embodiment, the polysaccharide may be sized to a lower molecular weight (MW) range than the molecular weight of the native capsular polysaccharide prior to conjugation with the carrier protein. The size of the purified capsular polysaccharide is reduced to produce a conjugate with advantageous filtration properties and / or yield.

[0133] In one such embodiment, the size of the purified capsular polysaccharide is reduced by high pressure homogenization, which achieves high shear rates by pumping the process stream through a channel with sufficiently small dimensions. The shear rate is increased by using a large applied homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0134] In one embodiment, the polysaccharides described herein are capable of inducing opsonic activity. In another embodiment, the polysaccharides described herein are capable of inducing opsonic and phagocytic activity (e.g., opsonophagocytic activity).

[0135] Opsonic activity or opsonization refers to the process by which an opsonin (e.g., an antibody or complement factor) binds to an antigen (e.g., an isolated polysaccharide described herein), which facilitates binding of the antigen to a phagocyte or phagocytic cell (e.g., macrophage, dendritic cell, and polymorphonuclear leukocyte (PMNL)). Some bacteria, such as encapsulated bacteria that are not typically phagocytosed due to the presence of a capsule, become more susceptible to recognition by phagocytes when coated with opsonic antibodies. In one embodiment, the polysaccharide elicits an immune response that is an opsonin, such as an antibody. In one embodiment, the opsonic activity is against gram-positive cocci, preferably against a Streptococcus species, more preferably against at least one strain of S. agalactiae.

[0136] In yet another embodiment, the polysaccharides described herein are capable of eliciting a bactericidal immune response, hi one embodiment, the bactericidal activity is against gram-positive cocci, preferably against Streptococcus species, more preferably against at least one strain of S. agalactiae.

[0137] Methods for measuring opsonization, phagocytosis and / or bactericidal activity are known in the art, for example, by measuring reduction in bacterial load in vivo (e.g., by measuring bacteremia levels in mammals inoculated with Streptococcus species) and / or by measuring bacterial cell killing in vitro (e.g., in vitro opsonophagocytosis assays). In one embodiment, the polysaccharide is capable of eliciting opsonization, phagocytosis and / or bactericidal activity compared to an appropriate control, such as, for example, compared to antisera raised against heat-killed Gram-positive cocci.

[0138] Serotype Ia One embodiment encompasses serotype Ia GBS capsular polysaccharide. The structure of serotype Ia can be depicted as follows:

[0139] [ka]

[0140] The molecular weight of the serotype Ia capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 100 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0141] In certain embodiments, a high pressure homogenization process is used to reduce the size of native GBS capsular polysaccharide serotype Ia while preserving structural features of the polysaccharide, such as sialic acid.

[0142] In one embodiment of the invention, the serotype Ia capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0143] In another embodiment, the serotype Ia capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0144] Serotype Ia capsular polysaccharide is less than about 5% O-acetylated. Some exemplary strains of serotype Ia capsular polysaccharide of the invention include 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524 and MB4052 (ATCC Accession No. 31574).

[0145] Serotype Ib One embodiment encompasses serotype Ib GBS capsular polysaccharide. The structure of serotype Ib can be depicted as follows:

[0146] [ka]

[0147] The molecular weight of the serotype Ib capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0148] In one embodiment of the invention, the serotype Ib capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0149] In another embodiment, the serotype Ib capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0150] Serotype Ib capsular polysaccharides are between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype Ib capsular polysaccharides of the invention include H36B (ATCC Accession No. 12401), S40, S42, MB4053 (ATCC Accession No. 31575), M709, 133, 7357 and PFEGBST0267.

[0151] Serotype II One embodiment encompasses serotype II GBS capsular polysaccharide. The structure of serotype II can be depicted as follows:

[0152] [ka]

[0153] The molecular weight of the serotype II capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0154] In one embodiment of the invention, the serotype II capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0155] In another embodiment, the serotype II capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0156] Serotype II capsular polysaccharides are less than about 5% O-acetylated. Some exemplary strains of serotype II capsular polysaccharides of the invention include MB4055 (ATCC Accession No. 31576), 18RS21 (ATCC Accession No. BAA-1175), S16, S20, V8 (ATCC Accession No. 12973), DK21, DK23, UAB, 5401 and PFEGBST0708.

[0157] Serotype III One embodiment encompasses serotype III GBS capsular polysaccharide. The structure of serotype III can be depicted as follows:

[0158] [ka]

[0159] The molecular weight of the serotype III capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Such as between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 100 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0160] In certain embodiments, a high pressure homogenization process is used to reduce the size of native GBS capsular polysaccharide serotype III while preserving structural features of the polysaccharide, such as sialic acid.

[0161] In one embodiment of the invention, the serotype III capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0162] In another embodiment, the serotype III capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0163] Serotype III capsular polysaccharides are between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype III capsular polysaccharides of the invention include MB4082 (ATCC Accession No. 31577), M132, 110, M781 (ATCC Accession No. BAA-22), D136C(3) (ATCC Accession No. 12403), M782, S23, 120, MB4316 (M-732; ATCC Accession No. 31475), M132, K79, COH1 (ATCC Accession No. BAA-1176) and PFEGBST0563.

[0164] Serotype IV One embodiment encompasses serotype IV GBS capsular polysaccharide. The structure of serotype IV can be depicted as follows:

[0165] [ka]

[0166] The molecular weight of the serotype IV capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0167] In one embodiment of the invention, the serotype IV capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0168] In another embodiment, the serotype IV capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0169] Serotype IV capsular polysaccharides are between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype IV capsular polysaccharides of the invention include 3139 (ATCC Accession No. 49446), CZ-NI-016, and PFEGBST0961.

[0170] Serotype V One embodiment encompasses serotype V GBS capsular polysaccharide. The structure of serotype V can be depicted as follows:

[0171] [ka]

[0172] The molecular weight of the serotype V capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 25 kDa and about 750 kDa, between about 25 kDa and about 500 kDa, between about 25 kDa and about 450 kDa, between about 25 kDa and about 400 kDa, between about 25 kDa and about 350 kDa, between about 25 kDa and about 300 kDa, between about 25 kDa and about 250 kDa, between about 25 kDa and about 200 kDa, between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 5 between about 0 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa,Between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is between about 25 kDa and about 400 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0173] In one embodiment of the invention, the serotype V capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0174] In another embodiment, the serotype V capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0175] Serotype V capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype V capsular polysaccharide of the invention include 1169-NT1, CJB111 (ATCC Accession No. BAA-23), CJB112, 2603V / R (ATCC Accession No. BAA-611), NCTC10 / 81, CJ11, and PFEGBST0837.

[0176] Serotype VI The GBS serotype VI capsular polysaccharide has been described by von Hunolstein, C. et al., Infection and Immunity, 6194):1272-1280 (1993), the disclosure of which is incorporated herein by reference in its entirety. The structure of serotype VI can be depicted as follows:

[0177] [ka]

[0178] The molecular weight of the serotype VI capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about between about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, about 100 kDa between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa Da, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0179] In one embodiment of the invention, the serotype VI capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0180] In another embodiment, the serotype VI capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0181] Serotype VI capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype VI capsular polysaccharide of the invention include 118754, 114852, 114862, 114866, 118775, B4589, B4645, SS1214 and CZ-PW-119.

[0182] Serotype VII The GBS serotype VII capsular polysaccharide has been described by Kogan, G. et al., Carbohydrate Research, 277(1):1-9 (1995), the disclosure of which is incorporated herein by reference in its entirety. The repeating unit of serotype VII is as follows:

[0183] [ka]

[0184] The molecular weight of the serotype VII capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about Between about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, between about 100 kDa and about 500 kDa, Da to about 400 kDa, between about 100 kDa to about 350 kDa, between about 100 kDa to about 300 kDa, between about 200 kDa to about 750 kDa, between about 200 kDa to about 700 kDa, between about 200 kDa to about 650 kDa, between about 200 kDa to about 600 kDa, between about 200 kDa to about 550 kDa, between about 200 kDa to about 500 kDa, between about 200 kDa to about 450 kDa, between about 200 kDa to about 400 kDa, between about 250 kDa to about 750 kDa, between about 250 kDa to about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0185] In one embodiment of the invention, the serotype VII capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0186] In another embodiment, the serotype VII capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0187] Serotype VII capsular polysaccharide is less than about 5% O-acetylated. Some exemplary strains of serotype VII capsular polysaccharide of the invention include 7271 and CZ-PW-045.

[0188] Serotype VIII The GBS serotype VIII capsular polysaccharide has been described by Kogan, G. et al., The Journal of Biological Chemistry, 271(15):8786-8790 (1996), the disclosure of which is incorporated herein by reference in its entirety. The repeating unit of serotype VIII is as follows:

[0189] [ka]

[0190] The molecular weight of the serotype VIII capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 50 ... between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 4 ... Da to about 400 kDa, between about 100 kDa to about 350 kDa, between about 100 kDa to about 300 kDa, between about 200 kDa to about 750 kDa, between about 200 kDa to about 700 kDa, between about 200 kDa to about 650 kDa, between about 200 kDa to about 600 kDa, between about 200 kDa to about 550 kDa, between about 200 kDa to about 500 kDa, between about 200 kDa to about 450 kDa, between about 200 kDa to about 400 kDa, between about 250 kDa to about 750 kDa, between about 250 kDa to about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0191] In one embodiment of the invention, the serotype VIII capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0192] In another embodiment, the serotype VIII capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0193] Serotype VIII capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype VIII capsular polysaccharide of the invention include JM9130013 and JM9130672.

[0194] Serotype IX The GBS serotype IX capsular polysaccharide has been previously described by Berti, F. et al., The Journal of Biological Chemistry, 289(34):23437-2348 (2014) and others. However, the configuration of the GlcpNAc in the backbone of the GBS serotype IX polysaccharide is alpha (α), which differs from previously published structural details that proposed this linkage to be in the β configuration. The structure of serotype IX can be more accurately depicted as follows:

[0195] [ka]

[0196] This structure, which corresponds to GBS serotype IX, can also be represented as follows:

[0197] [ka]

[0198] The molecular weight of the serotype IX capsular polysaccharide prior to conjugation may be between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa, between about 75 kDa and about 500 kDa, between about 75 kDa and about between about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 450 kDa, about 100 kDa between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about 700 kDa Da, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa, etc. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0199] In one embodiment of the invention, the serotype IX capsular polysaccharide comprises its native sialic acid level, such as about 100% or greater than about 95%. In another embodiment, the capsular polysaccharide may be desialylated prior to conjugation, such as up to about 40% (greater than about 60% sialylation level), such as up to about 35% (greater than about 65% sialylation level), up to about 30% (greater than about 70% sialylation level), up to about 25% (greater than about 75% sialylation level), up to about 20% (greater than about 80% sialylation level), up to about 15% (greater than about 85% sialylation level), up to about 10% (greater than about 90% sialylation level), or up to about 5% (greater than about 95% sialylation level).

[0200] In another embodiment, the serotype IX capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide, etc. In a further embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0.9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide, etc.

[0201] Serotype IX capsular polysaccharide is between about 0% and about 40% O-acetylated. In one embodiment of the invention, the polysaccharide is O-deacetylated (i.e., less than about 5% O-acetylated). Some exemplary strains of serotype IX capsular polysaccharide of the invention include IT-NI-016, IT-PW-62 and IT-PW-64.

[0202] Polysaccharide-protein conjugates As used herein, a "conjugate" comprises a capsular polysaccharide, usually of a desired molecular weight range, and a carrier protein, where the capsular polysaccharide is conjugated to the carrier protein. The conjugate may or may not contain some amount of free capsular polysaccharide. As used herein, "free capsular polysaccharide" refers to a capsular polysaccharide that is non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped within or by) the capsular polysaccharide-carrier protein that is conjugated. The terms "free capsular polysaccharide," "free polysaccharide," and "free saccharide" may be used interchangeably and are intended to convey the same meaning. Regardless of the nature of the carrier molecule, the carrier molecule can be conjugated to the capsular polysaccharide either directly or through a linker. As used herein, "conjugate," "conjugated," and "conjugating" refer to the process of covalently binding a bacterial capsular polysaccharide to a carrier molecule. Conjugation enhances the immunogenicity of bacterial capsular polysaccharides. Conjugation can be carried out according to the methods described below or by processes known in the art.

[0203] "Conjugate immunogenic composition" as used herein refers to an immunogenic composition in which the immunogenic material includes an antigenic polysaccharide covalently linked to a carrier protein to provide a polysaccharide-protein conjugate. In one embodiment, the polysaccharide-protein conjugates of the invention may be formulated as multivalent immunogenic compositions.

[0204] As used herein, the term "molecular weight" of a polysaccharide or of a carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0205] As used herein, "polysaccharide-protein conjugate" refers to a polysaccharide molecule conjugated to a protein carrier molecule through one or more covalent bonds. It may be desirable to conjugate the polysaccharide to a protein from another species known to be immunogenic in the target host. Thus, in one embodiment, the carrier molecule is a carrier protein. As defined herein, such a foreign protein is referred to as a "carrier protein". The carrier protein serves to enhance the antigenicity and immunogenicity of the polysaccharide. As used herein, the term "carrier effect" refers to the process in which the antigenicity and immunogenicity of a weakly immunogenic or non-immunogenic molecule is enhanced by being combined with a more immunogenic molecule (e.g., a heterologous protein) as a carrier. In this case, the polysaccharide in the combined polysaccharide-protein conjugate is more immunogenic than when presented alone. The carrier protein contains T cell epitopes to stimulate T cells that help generate an antibody response.

[0206] "Carrier protein" or "protein carrier", as used herein, refers to any protein molecule that can be conjugated with an antigen against which an immune response is desired (such as a capsular polysaccharide). Conjugation of an antigen, such as a polysaccharide, to a carrier protein can render the antigen immunogenic. Carrier proteins are preferably proteins that are non-toxic and non-reactogenic and obtainable in sufficient quantities and purity. Examples of carrier proteins are toxins, toxoids, or any mutant cross-reactive materials (CRMs) of toxins from tetanus, diphtheria, whooping cough, Pseudomonas species, E. coli, Staphylococcus species, and Streptococcus species. 197 ). The carrier protein must be amenable to standard conjugation procedures. In one embodiment, the carrier protein is Streptococcus C5a peptidase (SCP). In another embodiment of the invention, the CRM 197 is used as a carrier protein.

[0207] Cross-reactive materials or CRMs are particularly useful in some embodiments of the present invention. Genetically engineered proteins can be produced that are antigenically similar to certain bacterial toxins, but are non-toxic. These are called "cross-reactive materials" or CRMs. CRMs 197 (Wyeth / Pfizer Inc., Sanford, NC) is notable because it contains a single amino acid change from the native diphtheria toxin and is immunologically indistinguishable from it. See Pappenheimer, AM et al., Immunochem., 9(9):891-906 (1972); U.S. Patent No. 5,614,382, the disclosures of which are incorporated herein by reference in their entireties. CRM 197 CRM is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from cultures of Corynebacterium diphtheriae strain C7(β197) grown in a casamino acid and yeast extract-based medium. 197 CRM is purified via ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. 197 Cultures of C. diphtheriae strain C7(β197) which produce the protein have been deposited at the American Type Culture Collection, Rockville, Maryland, and have been assigned the accession number ATCC 53281. Other diphtheria toxoids are also suitable for use as carrier proteins. CRM3201 is a genetically engineered mutant of pertussis toxin. See Black, WJ et al., Science, 240(4852):656-659 (1988), the disclosure of which is incorporated herein by reference in its entirety.

[0208] Streptococcal C5a peptidase (SCP) is a cell wall-anchored virulence protein encoded by members of the genus beta-hemolytic Streptococcus that proteolytically inactivates the alpha fragment of complement component 5 (C5a), which is responsible for polymorphonuclear cell recruitment to the site of infection (2005.PNAS.102(51):18391.). It is a target for protective antibodies, and IgG antibodies directed against the SCP can mediate opsonophagocytosis. In addition, the SCP can serve as a carrier protein to enhance the immune response to the GBS CPS polysaccharide hapten to which it is conjugated.

[0209] Diphtheria toxoid (DT), CRM 197In addition to SCP and pertussis toxoid, further examples of carrier proteins include tetanus toxoid (TT), cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004 / 083251), E. coli heat labile toxoid (LT), E. coli heat stable toxoid (ST), pneumolysin from S. pneumoniae (wild type or a virulence-reduced mutant), pneumococcal surface protein A (PspA), pneumococcal adhesin protein A (PsaA), C5a peptidase from Streptococcus, hemolysin from Staphylococcal aureus, nontypeable Haemophilus influenzae (NTHi) protein, Haemophilus influenzae protein D, Clostridium perfringens (Clostridium perfringens) and the like. Examples of exotoxins include P. perfringens exotoxin / toxoid, Hepatitis B surface antigen, Hepatitis B core antigen, Rotavirus VP7 protein, and respiratory syncytial virus F and G proteins, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derivative (PPD) of tuberculin, and Pseudomonas exotoxin or derivatives thereof, including recombinantly produced non-toxic mutant Pseudomonas aeruginosa exotoxin A. Bacterial outer membrane proteins, such as outer membrane protein complex c (OMPC), porins, transferrin-binding proteins, etc., or C. difficile enterotoxin (toxin A) and cytotoxin (toxin B) can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), can also be used as carrier proteins. In a preferred embodiment, the carrier protein is diphtheria toxoid. More preferably, the carrier protein is CRM 197 In another embodiment of the invention, the carrier protein is tetanus toxoid.

[0210] For the synthesis of a multivalent conjugate immunogenic composition, a polysaccharide-protein conjugate can be produced by conjugating a mixture of polysaccharides purified from two different species of bacteria with a carrier protein. Alternatively, a multivalent conjugate immunogenic composition can be produced by combining polysaccharides purified from two or more different serotypes of the same bacterium and conjugating them as a mixture with a carrier protein. Alternatively, polysaccharide-protein conjugates produced by reacting a single type of polysaccharide with a carrier protein in separate reactions using different polysaccharides can be mixed. Thus, a multivalent immunogenic composition can include a carrier protein carrying a homogeneous or heterogeneous population of bound polysaccharides.

[0211] After conjugation of the capsular polysaccharide with the carrier protein, the polysaccharide-protein conjugate is purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various techniques, including, for example, concentration / hemodiafiltration operations, precipitation / elution, column chromatography and depth filtration.

[0212] As mentioned above, the present invention relates to a conjugate comprising a GBS capsular polysaccharide conjugated to a carrier protein. One embodiment of the present invention provides a conjugate comprising a GBS serotype VI capsular polysaccharide conjugated to a carrier protein and at least one further conjugate comprising a GBS serotype Ia capsular polysaccharide conjugated to a carrier protein, a GBS serotype Ib capsular polysaccharide conjugated to a carrier protein, a GBS serotype II capsular polysaccharide conjugated to a carrier protein, a GBS serotype III capsular polysaccharide conjugated to a carrier protein, a GBS serotype V capsular polysaccharide conjugated to a carrier protein, a GBS serotype VII capsular polysaccharide conjugated to a carrier protein, a GBS serotype VIII capsular polysaccharide conjugated to a carrier protein, or a GBS serotype IX capsular polysaccharide conjugated to a carrier protein. In one aspect of the invention, the polysaccharide has a molecular weight between about 5 kDa and 1,000 kDa, the conjugate has a molecular weight between about 300 kDa and about 20,000 kDa, and the conjugate comprises less than about 40% free polysaccharide relative to the total polysaccharide, hi one embodiment, the conjugate comprises less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free polysaccharide relative to the total polysaccharide.

[0213] In one embodiment, the serotype Ia, Ib, II, III, IV, V, VI, VII, VIII and / or IX polysaccharides are, prior to conjugation, between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 750 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa and about 450 kDa, between about 50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa, between about 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa, between about 50 kDa and about 200 kDa, between about 75 kDa and about 750 kDa. between about 75 kDa and about 500 kDa, between about 75 kDa and about 450 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 350 kDa, between about 75 kDa and about 300 kDa, between about 75 kDa and about 250 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 750 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 650 kDa, between about 100 kDa and about 600 kDa, between about 100 kDa and about 550 kDa, between about 100 kDa and about 500 kDa, about 100 kDa between about 100 kDa and about 400 kDa, between about 100 kDa and about 350 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 750 kDa, between about 200 kDa and about 700 kDa, between about 200 kDa and about 650 kDa, between about 200 kDa and about 600 kDa, between about 200 kDa and about 550 kDa, between about 200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa, between about 200 kDa and about 400 kDa, between about 250 kDa and about 750 kDa, between about 250 kDa and about and / or about 700 kDa, between about 250 kDa and about 650 kDa, between about 250 kDa and about 600 kDa, between about 250 kDa and about 550 kDa, between about 250 kDa and about 500 kDa, between about 250 kDa and about 450 kDa, between about 250 kDa and about 400 kDa, between about 300 kDa and about 750 kDa, between about 300 kDa and about 700 kDa, between about 300 kDa and about 650 kDa, between about 300 kDa and about 600 kDa, between about 300 kDa and about 550 kDa, or between about 300 kDa and about 500 kDa.Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0214] In one embodiment, the conjugate is between about 300 kDa and about 20,000 kDa, e.g., between about 300 kDa and about 15,000 kDa, between about 300 kDa and about 10,000 kDa, between about 300 kDa and about 9,000 kDa, between about 300 kDa and about 8,000 kDa, between about 300 kDa and about 7,000 kDa, between about 300 kDa and about 6,000 kDa, between about 300 kDa and about 5,000 kDa, between about 300 kDa and about 4,000 kDa, between about 300 kDa and about 3,000 kDa, between about 300 kDa and about 2, 000 kDa, between about 300 kDa and about 1,000 kDa, between about 500 kDa and about 20,000 kDa, between about 500 kDa and about 15,000 kDa, between about 500 kDa and about 10,000 kDa, between about 500 kDa and about 9,000 kDa, between about 500 kDa and about 8,000 kDa, between about 500 kDa and about 7,000 kDa, between about 500 kDa and about 6,000 kDa, between about 500 kDa and about 5,000 kDa, between about 500 kDa and about 4,000 kDa, between about 500 kDa and about 3,000 kDa, between about 500 kDa and about 5,000 kDa Da to about 2,000 kDa, between about 500 kDa to about 1,000 kDa, between about 1,000 kDa to about 20,000 kDa, between about 1,000 kDa to about 15,000 kDa, between about 1,000 kDa to about 10,000 kDa, between about 1,000 kDa to about 9,000 kDa, between about 1,000 kDa to about 8,000 kDa, between about 1,000 kDa to about 7,000 kDa, between about 1,000 kDa to about 6,000 kDa, between about 1,000 kDa to about 5,000 kDa, between about 1,500 kDa to about 20,000 kDa, between about 1,500 kDa and about 15,000 kDa, between about 1,500 kDa and about 10,000 kDa, between about 1,500 kDa and about 9,000 kDa, between about 1,500 kDa and about 8,000 kDa, between about 1,500 kDa and about 7,000 kDa, between about 1,500 kDa and about 6,000 kDa, between about 1,500 kDa and about 5,000 kDa, between about 2,000 kDa and about 20,000 kDa, between about 2,000 kDa and about 15,000 kDa, between about 2,000 kDa and about 10,000 kDa, between about 2,000 kDa and about 9,between about 2,000 kDa and about 8,000 kDa, between about 2,000 kDa and about 7,000 kDa, between about 2,000 kDa and about 6,000 kDa, between about 2,500 kDa and about 20,000 kDa, between about 2,500 kDa and about 15,000 kDa, between about 2,500 kDa and about 10,000 kDa, between about 2,500 kDa and about 9,000 kDa, between about 2,500 kDa and about 8,000 kDa, between about 2,500 kDa and about 7 For example, the ribozyme may have a molecular weight of between about 2,000 kDa, between about 2,500 kDa and about 6,000 kDa, between about 3,000 kDa and about 20,000 kDa, between about 3,000 kDa and about 15,000 kDa, between about 3,000 kDa and about 10,000 kDa, between about 3,000 kDa and about 9,000 kDa, between about 3,000 kDa and about 8,000 kDa, between about 3,000 kDa and about 7,000 kDa, or between about 3,000 kDa and about 6,000 kDa.

[0215] In certain embodiments, the GBS serotype VI capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0216] In certain embodiments, the GBS serotype Ia capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0217] In certain embodiments, the GBS serotype Ib capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0218] In certain embodiments, the GBS serotype II capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0219] In certain embodiments, the GBS serotype III capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0220] In certain embodiments, the GBS serotype V capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0221] In certain embodiments, the GBS serotype VII capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0222] In certain embodiments, the GBS serotype VIII capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0223] In certain embodiments, the GBS serotype IX capsular polysaccharide conjugate has a molecular weight within any of the above ranges.

[0224] In one embodiment, the conjugates of the invention have at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.97 or 0.98 mM sialic acid per mM polysaccharide. In a preferred embodiment, the conjugates have at least about 0.9 or 0.95 mM sialic acid per mM polysaccharide.

[0225] In certain embodiments, the GBS serotype VI capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0226] In certain embodiments, the GBS serotype Ia capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0227] In certain embodiments, the GBS serotype Ib capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0228] In certain embodiments, the GBS serotype II capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0229] In certain embodiments, the GBS serotype III capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0230] In certain embodiments, the GBS serotype V capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0231] In certain embodiments, the GBS serotype VII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0232] In certain embodiments, the GBS serotype VIII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0233] In certain embodiments, the GBS serotype IX capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

[0234] In certain embodiments, the conjugates of the invention comprise less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM repeating saccharide unit, hi other embodiments, the conjugates comprise at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM O-acetate per mM repeating saccharide unit.

[0235] In certain embodiments, the GBS serotype VI capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0236] In certain embodiments, the GBS serotype Ia capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0237] In certain embodiments, the GBS serotype Ib capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0238] In certain embodiments, the GBS serotype II capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0239] In certain embodiments, the GBS serotype III capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0240] In certain embodiments, the GBS serotype V capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0241] In certain embodiments, the GBS serotype VII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0242] In certain embodiments, the GBS serotype VIII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0243] In certain embodiments, the GBS serotype IX capsular polysaccharide conjugate has an O-acetate content of any of the above values.

[0244] In further embodiments, the immunogenic conjugate comprises less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free GBS capsular polysaccharide relative to the total amount of GBS capsular polysaccharide. In a preferred embodiment, the immunogenic conjugate comprises less than about 5% unreacted free saccharide relative to the total amount of GBS capsular polysaccharide.

[0245] In yet another embodiment, the ratio of GBS capsular polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.0. In one aspect, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.5 and about 2.0, between about 0.5 and about 1.5, between about 0.5 and about 1.0, between about 1.0 and about 1.5, or between about 1.0 and about 2.0. In a preferred embodiment, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.8 and about 1.0.

[0246] In another embodiment, the degree of conjugation of the conjugate is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15, or between 10 and 12. In a preferred embodiment, the degree of conjugation of the conjugate is between 2 and 5.

[0247] Conjugation Conjugation can be direct, where an atom from the polysaccharide is covalently bonded to an atom from the protein surface, or alternatively, conjugation can be via a linker molecule, which reacts with both the polysaccharide and the protein and connects the two, tethering the carbohydrate to the protein.

[0248] When the carrier and one or more antigens, such as polysaccharides, are conjugated (i.e., covalently associated), the conjugation can be by any chemical method, process, or genetic technique known to those of skill in the art. For example, the carrier polypeptide and one or more antigens selected from the group including carbohydrates, oligosaccharides, lipids, lipooligosaccharides, polysaccharides, oligosaccharide-protein conjugates, polysaccharide-protein conjugates, peptide-protein conjugates, oligosaccharide-peptide conjugates, polysaccharide-peptide conjugates, protein-protein conjugates, lipooligosaccharide-protein conjugates, polysaccharide-protein conjugates, or any combination thereof, can be coupled to the carrier polypeptide by (1) direct coupling through protein functional groups (e.g., thiol-thiol bonds, amine-carboxyl bonds, amine-aldehyde bonds; enzymatic direct coupling), (2) homobifunctional coupling of amines (e.g., using bis-aldehydes), (3) homobifunctional coupling of thiols (e.g., using bis-maleimides), (4) direct coupling of thiols (e.g., using bis-maleimides), (5) direct coupling of thiols (e.g., using bis-maleimides), (6) direct coupling of thiols (e.g., using bis-maleimides), (7) direct coupling of thiols (e.g., using bis-maleimides), (8) direct coupling of thiols (e.g., using bis-maleimides), (9) direct coupling of thiols (e.g., using bis-maleimides), (10) direct coupling of thiols (e.g., using bis-maleimides), (11) direct coupling of thiols (e.g., using bis-maleimides), (12) direct coupling of thiols (e.g., using bis-maleimides), (13) direct coupling of thiols (e.g., using bis-maleimides), (4) homobifunctional coupling via photoactivatable reagents, (5) heterobifunctional coupling of amines with thiols (e.g., using maleimides), (6) heterobifunctional coupling via photoactivatable reagents (e.g., the β-carbonyidiazo family), (7) introduction of amine-reactive groups into polysaccharides or oligosaccharides via cyanogen bromide activation or carboxymethylation, (8) introduction of thiol-reactive groups into polysaccharides or oligosaccharides via heterobifunctional compounds such as maleimide-hydrazides, (9) protein-lipid conjugation via introduction of hydrophobic groups into proteins, and (10) protein-lipid conjugation via incorporation of reactive groups into lipids. Heterobifunctional "non-covalent coupling" techniques such as biotin-avidin interactions are also contemplated. Other methods known in the art for achieving conjugation of oligo- and polysaccharides to immunogenic carrier proteins are also within the scope of some embodiments of the present invention.

[0249] In one embodiment, the GBS capsular polysaccharide-protein conjugate is obtained by activating the polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide can be coupled to an amino group on a carrier protein directly or via a spacer (linker) group. For example, the spacer can be cystamine or cysteamine to obtain a thiolated polysaccharide that can be coupled to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using GMBS) or a haloacetylated carrier protein (e.g., using iodoacetimide, SIB, SIAB, sulfo-SIAB, SIA or SBAP).

[0250] In one embodiment, a cyanate ester (which may be made by CDAP chemistry) is coupled with hexanediamine or adipic dihydrazide (ADH), and an amino-derivatized sugar is conjugated to a carrier protein using carbodiimide (e.g., EDAC or EDC) chemistry via carboxyl groups on the protein carrier. Such conjugates are described, for example, in International Patent Application Publication Nos. WO93 / 15760, WO95 / 08348, and WO96 / 29094.

[0251] Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC and TSTU. Many are described in International Patent Application Publication No. WO98 / 42721. Conjugation may involve a carbonyl linker that can be formed by reaction of the free hydroxyl group of the sugar with 1,1 carbonyldiimidazole (CDI) or 1,1 carbonyldi-1,2,4 triazole (CDT) (see Bethell et al., J. Biol. Chem., 254:2572-2574 (1979); Hearn et al., J. Chromatogr., 218:509-518 (1981)), followed by reaction with the protein to form a carbamate bond. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI / CDT to form a CDI / CDT carbamate intermediate, and coupling the CDI / CDT carbamate intermediate to an amino group on the protein.

[0252] In a preferred embodiment, the GBS capsular polysaccharide-protein conjugates of the invention are prepared using reductive amination, which involves two steps: (1) oxidation of the polysaccharide to generate aldehyde functional groups from vicinal diols in individual hexasaccharide units, and (2) reduction of the activated polysaccharide and carrier protein to form the conjugate.

[0253] In one embodiment, the GBS capsular polysaccharide is (a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction by the addition of a quenching agent to provide activated GBS capsular polysaccharide. It is activated (oxidized) by a process that involves

[0254] In certain embodiments of the invention, the concentration of the isolated capsular polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL, such as between about 0.5 mg / mL and about 5.0 mg / mL, between about 1.0 mg / mL and about 3.0 mg / mL, or about 2.0 mg / mL.

[0255] In certain embodiments, the oxidizing agent is periodate. Periodate oxidizes adjacent hydroxyl groups to form carbonyl or aldehyde groups, resulting in the cleavage of the C-C bond. The term "periodate" encompasses both periodate and periodic acid. The term also includes metaperiodate (IO4 - ) and orthoperiodate (IO6 5- The term "periodate" also encompasses both periodate and potassium periodate. The term "periodate" also encompasses various salts of periodate, including sodium periodate and potassium periodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In a preferred embodiment, the periodate used to oxidize GBS capsular polysaccharide is metaperiodate. In a preferred embodiment, the periodate used to oxidize serotype capsular polysaccharide is sodium metaperiodate.

[0256] In another embodiment, the polysaccharide is reacted with 0.01 to 10.0, 0.05 to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7 to 0.8, 0.05 to 0.5, or 0.1 to 0.3 molar equivalents of the oxidizing agent. In certain embodiments, the polysaccharide is reacted with about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95 molar equivalents of the oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.1 molar equivalents of the oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.15 molar equivalents of the oxidizing agent. In an additional embodiment, the polysaccharide is reacted with about 0.25 molar equivalents of the oxidizing agent. In yet another embodiment, the polysaccharide is reacted with about 0.5 molar equivalents of the oxidizing agent. In an alternative embodiment, the polysaccharide is reacted with about 0.6 molar equivalents of the oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.7 molar equivalents of the oxidizing agent.

[0257] In one embodiment of the invention, the duration of the oxidation reaction is between about 1 hour and about 50 hours, between about 10 hours and about 30 hours, between about 15 hours and about 20 hours, between about 15 hours and about 17 hours, or about 16 hours.

[0258] In another aspect of the invention, the temperature of the oxidation reaction is maintained between about 2° C. and about 25° C., between about 2° C. and about 8° C., or between about 20° C. and about 25° C. In one preferred embodiment, the temperature of the reaction is maintained at about 23° C. In another preferred embodiment, the temperature of the reaction is maintained at about 5° C.

[0259] In a further aspect, the oxidation reaction is carried out in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES) and Bis-Tris. In a preferred embodiment, the buffer is potassium phosphate.

[0260] In additional embodiments, the buffer has a concentration of between about 1 mM and about 500 mM, between about 1 mM and about 300 mM, or between about 50 mM and about 200 mM. In a preferred embodiment, the buffer has a concentration of about 100 mM.

[0261] In one aspect, the oxidation reaction is carried out at a pH between about 4.0 and about 8.0, between about 5.0 and about 7.0, or between about 5.5 and about 6.5. In a preferred embodiment, the pH is about 6.0.

[0262] In one embodiment, the activated GBS capsular polysaccharide is obtained by reacting about 0.5 mg / L to about 5.0 mg / mL of isolated capsular polysaccharide with about 0.05 to about 0.3 molar equivalents of periodate at a temperature between about 20° C. and 25° C.

[0263] In another embodiment, the activated GBS capsular polysaccharide is obtained by reacting about 0.5 mg / L to about 5.0 mg / mL of isolated capsular polysaccharide with about 0.05 to about 0.3 molar equivalents of periodate at a temperature between about 2° C. and about 8° C.

[0264] In another embodiment, the activated GBS capsular polysaccharide is purified according to methods known to those skilled in the art, such as gel permeation chromatography (GPC), dialysis or ultrafiltration / hemodiafiltration, etc. For example, the activated capsular polysaccharide is purified by hemodiafiltration using a concentration and ultrafiltration device.

[0265] In one embodiment, the degree of oxidation of the activated GBS capsular polysaccharide is between 5 and 25, such as between 5 and 15, between 5 and 10, between 10 and 25, between 10 and 20, between 10 and 15, etc. In a preferred embodiment, the degree of oxidation of the activated GBS capsular polysaccharide is between 10 and 20, between 11 and 19, between 12 and 18, between 13 and 17, or between 14 and 16.

[0266] In another embodiment, the activated GBS capsular polysaccharide has a molecular weight between about 5 kDa and about 1,000 kDa, e.g., between about 50 kDa and about 300 kDa, between about 75 kDa and about 400 kDa, between about 75 kDa and about 200 kDa, between about 100 kDa and about 700 kDa, between about 100 kDa and about 500 kDa, between about 100 kDa and about 400 kDa, between about 100 kDa and about 300 kDa, between about 200 kDa and about 400 kDa, between about 300 kDa and about 700 kDa, etc. In a preferred embodiment, the activated GBS capsular polysaccharide has a molecular weight between about 75 kDa and about 400 kDa.

[0267] In one embodiment, the activated GBS capsular polysaccharide is lyophilized, optionally in the presence of a sugar. In a preferred embodiment, the sugar is selected from sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit. In a preferred embodiment, the sugar is sucrose. The lyophilized activated capsular polysaccharide can then be combined with a solution containing a carrier protein.

[0268] In another embodiment, the activated GBS capsular polysaccharide is combined with a carrier protein and lyophilized, optionally in the presence of a sugar. In one aspect, the sugar is selected from sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit. In a preferred embodiment, the sugar is sucrose. The co-lyophilized polysaccharide and carrier protein can then be resuspended in a solution and reacted with a reducing agent.

[0269] Activated GBS capsular polysaccharides are (a) combining activated GBS capsular polysaccharide with a carrier protein; (b) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; The antibody can be conjugated to a carrier protein by a process comprising:

[0270] Conjugation of activated GBS capsular polysaccharide to a protein carrier by reductive amination in a polar aprotic solvent is suitable for maintaining low levels of free polysaccharide, as compared, for example, to reductive amination in aqueous solution, where the level of unreacted (free) polysaccharide increases significantly. In a preferred embodiment, steps (a) and (b) are carried out in a polar aprotic solvent.

[0271] In one embodiment, step (a) comprises dissolving the lyophilized GBS capsular polysaccharide in a solution comprising the carrier protein and a polar aprotic solvent, hi another embodiment, step (a) comprises dissolving the co-lyophilized GBS capsular polysaccharide and carrier protein in a polar aprotic solvent.

[0272] In one embodiment, the polar aprotic solvent is selected from the group consisting of dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF) and hexamethylphosphoramide (HMPA). In a preferred embodiment, the polar aprotic solvent is DMSO.

[0273] When steps (a) and (b) are carried out in an aqueous solution, steps (a) and (b) are preferably carried out in a buffer in the aqueous medium at a pH between about 6.0 and about 8.5, between about 7.0 and about 8.0, or between about 7.0 and about 7.5, selected from PBS, MES, HEPES, Bis-Tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine, or HEPB. In a preferred embodiment, the buffer is PBS. In a preferred embodiment, the pH is about 7.3.

[0274] In one embodiment, the concentration of activated GBS capsular polysaccharide in step (b) is between about 0.1 mg / mL and about 10.0 mg / mL, between about 0.5 mg / mL and about 5.0 mg / mL, or between about 0.5 mg / mL and about 2.0 mg / mL. In a preferred embodiment, the concentration of activated serotype GBS capsular polysaccharide in step (b) is between 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, about 3.0 mg / mL, about 3.1 mg / mL, about 3.2 mg / mL, about 3.4 mg / mL, about 3.5 mg / mL, about 3.6 mg / mL, about 3.7 mg / mL, about 3.8 mg / mL, about 3.9 mg / mL, about 3.8 mg / mL, about 3.9 mg / mL, about 3.1 mg / mL, about 3.2 mg / mL, about 3.3 mg / mL, about 3.4 mg / mL, about 3.5 mg / mL 0.4 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.

[0275] In another embodiment, the initial ratio (weight / weight) of activated serotype GBS capsular polysaccharide to carrier protein is between 5:1 and 0.1:1, 2:1 and 0.1:1, 2:1 and 1:1, 1.5:1 and 1:1, 0.1:1 and 1:1, 0.3:1 and 1:1, 0.6:1 and 1:1. In a preferred embodiment, the initial ratio of activated serotype GBS capsular polysaccharide to carrier protein is about 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1.

[0276] In certain embodiments, the reducing agent is a Bronsted or Lewis acid, an amine borane, such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe iThe reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc in the presence of PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB), etc. In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0277] In another embodiment, the amount of reducing agent used in step (b) is between about 0.1 and about 10.0 molar equivalents, between about 0.5 and about 5.0 molar equivalents, or between about 1.0 and about 2.0 molar equivalents. In a preferred embodiment, the amount of reducing agent used in step (b) is about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 molar equivalents.

[0278] In yet another embodiment, the duration of step (b) is between 1 hour and 60 hours, between 10 hours and 50 hours, between 40 hours and 50 hours, or between 42 hours and 46 hours. In a preferred embodiment, the duration of step (b) is about 44 hours.

[0279] In further embodiments, the temperature of the reaction in step (b) is maintained between 10° C. and 40° C., between 15° C. and 30° C., or between 20° C. and 26° C. In a preferred embodiment, the temperature of the reaction in step (b) is maintained at about 23° C.

[0280] In an additional embodiment, the process for the preparation of an immunogenic conjugate comprising GBS capsular polysaccharide covalently bound to a carrier protein further comprises a step of capping (quenching) unreacted aldehydes by the addition of borohydride (step (c)).

[0281] In one embodiment, the capping reagent is a borohydride selected from the group consisting of sodium borohydride (NaBH4), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride and magnesium borohydride. In a preferred embodiment, the capping reagent is sodium borohydride.

[0282] In yet another embodiment, the amount of borohydride used in step (c) is between about 0.1 and about 10.0 molar equivalents, between about 0.5 and about 5.0 molar equivalents, or between about 1.0 and 3.0 molar equivalents. In a preferred embodiment, the amount of borohydride used in step (c) is about 2.0 molar equivalents.

[0283] In one embodiment, the borohydride used in step (c) is NaBH4 at a concentration of about 2.0 molar equivalents.

[0284] In one embodiment, the duration of step (c) is between 0.1 and 10 hours, between 0.5 and 5 hours, between 2 and 4 hours. In a preferred embodiment, the duration of step (c) is about 3 hours.

[0285] In another embodiment, the temperature of the reaction in step (c) is maintained between about 15° C. and about 45° C., between about 15° C. and about 30° C., or between about 20° C. and about 26° C. In a preferred embodiment, the temperature of the reaction in step (c) is maintained at about 23° C.

[0286] After conjugation and capping of the GBS capsular polysaccharide with the carrier protein, the polysaccharide-protein conjugate can be purified (enriched in terms of the amount of polysaccharide-protein conjugate) by a variety of techniques known to those skilled in the art, including dialysis, concentration / hemodiafiltration operations, tangential flow filtration, precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography) and depth filtration.

[0287] In further embodiments, the immunogenic conjugate comprises less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free GBS capsular polysaccharide relative to the total amount of GBS capsular polysaccharide. In a preferred embodiment, the immunogenic conjugate comprises less than about 5% unreacted free saccharide relative to the total amount of GBS capsular polysaccharide.

[0288] In another embodiment, the GBS polysaccharide-protein conjugate has a molecular weight between about 300 kDa and about 20,000 kDa, such as between about 1,000 kDa and about 15,000 kDa or between about 1,000 kDa and about 10,000 kDa.

[0289] In yet another embodiment, the ratio of GBS capsular polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.0. In one aspect, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.5 and about 2.0, between about 0.5 and about 1.5, between about 0.5 and about 1.0, between about 1.0 and about 1.5, or between about 1.0 and about 2.0. In a preferred embodiment, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is between about 0.8 and about 1.0.

[0290] In another embodiment, the degree of conjugation of the conjugate is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15, or between 10 and 12. In a preferred embodiment, the degree of conjugation of the conjugate is between 2 and 5.

[0291] In one embodiment of the present invention, the GBS capsular polysaccharide-protein conjugate is obtained by the reductive amination method described above. For example, in one embodiment, the present disclosure provides a method for the preparation of a GBS capsular polysaccharide-protein conjugate comprising: (a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction by the addition of a quenching agent to provide activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; and optionally (e) capping any unreacted aldehyde by addition of sodium borohydride (NaBH4); The present invention provides a GBS capsular polysaccharide-protein conjugate comprising a polysaccharide conjugated to a carrier protein, the polysaccharide being produced or obtainable by a method comprising the steps of:

[0292] In a preferred embodiment, steps (c) and (d) are carried out in DMSO.

[0293] In another aspect of the invention, the GBS capsular polysaccharide-protein conjugates of the invention are prepared using reductive amination as described above, except that 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) are used as co-oxidants in the activation / oxidation step. See International Patent Application Publication No. WO2014 / 097099, which is incorporated by reference in its entirety. In such an embodiment, the GBS capsular polysaccharide-derived glycoconjugates are prepared using TEMPO free radical to oxidize the primary alcohol of the sugar to an aldehyde using NCS as a co-oxidant (hereinafter "TEMPO / NCS oxidation"), as described in Example 7 and in International Patent Application Publication No. WO2014 / 097099. Thus, in one aspect, conjugates of GBS capsular polysaccharide are obtainable by a method comprising the steps of a) reacting GBS capsular polysaccharide with TEMPO and NCS in a solvent to produce an activated saccharide, and b) reacting the activated saccharide with a carrier protein containing one or more amine groups (hereinafter "TEMPO / NCS-reductive amination"). In one embodiment, the solvent may be an aqueous solvent or DMSO.

[0294] In one embodiment, the GBS capsular polysaccharide-protein conjugate is obtained by the method. For example, in one embodiment, the present disclosure provides a GBS capsular polysaccharide-protein conjugate comprising a polysaccharide conjugated to a carrier protein, which is produced or obtainable by a method comprising: a) reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in a solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amine groups. In one embodiment, the solvent may be an aqueous solvent or DMSO.

[0295] immunogenic composition After the individual conjugates are purified, they can be combined to formulate an immunogenic composition of the invention that can be used, for example, in a vaccine. Formulation of the immunogenic composition of the invention can be accomplished using art-recognized methods.

[0296] An "immune response" to an immunogenic composition is the development in a subject of a humoral and / or cell-mediated immune response to molecules (e.g., antigens such as proteins or polysaccharides) present in the composition of interest. For the purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response, involving the production of antibodies with affinity for antigens present in the immunogenic composition of the present invention, whereas a "cell-mediated immune response" is one mediated by T-lymphocytes and / or other white blood cells. A "cell-mediated immune response" is elicited by the presentation of antigen epitopes in association with class I or class II molecules of the major histocompatibility complex (MHC). This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic T lymphocyte cells (CTLs). CTLs have specificity for peptide or lipid antigens that are presented in association with proteins encoded by MHC or CD1 and expressed on the surface of cells. CTLs help induce and promote the intracellular destruction of intracellular microorganisms, or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves antigen-specific responses by helper T cells. Helper T cells act to stimulate function and help focus the activity of non-specific effector cells against cells displaying peptide antigens in association with classical or non-classical MHC molecules on their surface. "Cell-mediated immune response" also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other white blood cells, including those derived from CD4+ and CD8+ T cells. The ability of a particular antigen or composition to stimulate a cell-mediated immunological response can be determined by a number of assays, such as by lymphocyte proliferation (lymphocyte activation) assays, CTL cytotoxicity cell assays, by assaying for T-lymphocytes specific for the antigen in sensitized subjects, or by measuring cytokine production by T cells in response to restimulation with antigen. Such assays are well known in the art.See, e.g., Erickson, A.L. et al., J. Immunol., 151(8):4189-4199 (1993); Doe, B. et al., Eur. J. Immunol., 24(10):2369-2376 (1994).

[0297] The term "immunogenicity" refers to the ability of an antigen or vaccine to elicit either a humoral or cell-mediated immune response, or both.

[0298] An "immunogenic amount" or an "immunologically effective amount" or a "dose", each of which are used interchangeably herein, generally refer to an amount of an antigen or immunogenic composition sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, or both, as measured by standard assays known to those of skill in the art.

[0299] "Immune interference" or "significant immune interference", as used herein, refers to a statistically significant reduction in the immune response to an individual antigen in a multivalent or multicomponent vaccine compared to the immune response to the same antigen when administered in a monovalent vaccine.

[0300] A "protective" immune response refers to the ability of an immunogenic composition to elicit an immune response, either humoral or cell-mediated, that serves to protect a subject from infection. The protection provided need not be absolute, i.e., to completely prevent or eradicate infection, provided there is a statistically significant improvement compared to a control population of subjects, e.g., infected animals that have not been administered the vaccine or the immunogenic composition. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of infection. Several assays are known in the art for determining whether an immune response is indicative of a "protective immune response." For example, an increase in antibody levels may be measured by a binding assay, such as a whole cell ELISA assay, as described further below. Other assays include measuring functional antibody responses, such as the facilitation of bacterial killing, which may be tested with an opsonophagocytosis assay (OPA), as described below. In certain circumstances, a "protective immune response" may include eliciting a two-fold increase in antibody levels or a four-fold increase in antibody levels specific to a particular antigen in at least 50% of subjects. In other contexts, a "protective immune response" can encompass a reduction in bacterial counts of at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more.

[0301] The amount of a particular conjugate in a composition is generally calculated based on the total conjugated and unconjugated polysaccharide for that conjugate. For example, a GBS capsular polysaccharide conjugate with 20% free polysaccharide would be expected to have about 80mcg / ml of conjugated GBS capsular polysaccharide and about 20mcg / ml of unconjugated GBS capsular polysaccharide in a 100mcg / ml GBS capsular polysaccharide dose. The contribution of the protein carrier to the conjugate is not usually taken into account when calculating the dose of the conjugate. The amount of conjugate may vary depending on the serotype of the streptococcus. Generally, each dose will contain about 0.01mg / ml to about 100mcg / ml of each polysaccharide, particularly about 1mcg / ml to about 70mcg / ml, more preferably about 5mcg / ml to about 50mcg / ml. The "immunogenic amounts" of the different polysaccharide components in the immunogenic composition may vary, with each amount being about 0.01 mcg / ml, about 0.1 mcg / ml, about 0.25 mcg / ml, about 0.5 mcg / ml, about 1 mcg / ml, about 2 mcg / ml, about 3 mcg / ml, about 4 mcg / ml, about 5 mcg / ml, about 6 mcg / ml, about 7 mcg / ml, about 8 mcg / ml, about 9 mcg / ml, about 10 mcg / ml, about 11 mcg / ml, about 12 mcg / ml, about 13 mcg / ml, about 14 mcg / ml, about 15 mcg / ml, about 16 mcg / ml, about 17 mcg / ml, about 18 mcg / ml, about 19 mcg / ml, about 20 The polysaccharide antigen may comprise about 100mcg / ml, about 9mcg / ml, about 10mcg / ml, about 15mcg / ml, about 20mcg / ml, about 25mcg / ml, about 30mcg / ml, about 40mcg / ml, about 50mcg / ml, about 60mcg / ml, about 70mcg / ml, about 80mcg / ml, about 90mcg / ml, or about 100mcg / ml of any particular polysaccharide antigen. A dose or immunogenic amount of a multivalent immunogenic composition is expected to indicate the dose of each polysaccharide unless otherwise indicated. For example, a 10mcg / ml dose of a hexavalent immunogenic composition is expected to contain 10mcg / ml of each of the six polysaccharides.

[0302] The effectiveness of an antigen as an immunogen can be measured by measuring the level of circulating antibodies specific for the antigen in serum, by measuring the level of B cell activity, using immunoassays, immunoprecipitation assays, functional antibody assays, such as in vitro opsonization assays, and many other assays known in the art. Another measure of the effectiveness of an antigen as a T cell immunogen can be measured either by proliferation assays, by cytolytic assays, such as chromium release assays to measure the ability of T cells to lyse their specific target cells, and the like. Furthermore, in the present invention, an "immunogenic amount" may be defined by measuring the serum level of antigen-specific antibodies elicited after administration of the antigen, or by measuring the ability of the antibodies so elicited to enhance the opsonophagocytic ability of certain leukocytes as described herein. The level of protection of the immune response can be measured by inoculating an immunized host with the injected antigen. For example, if the antigen against which an immune response is desired is a bacterium, the level of protection elicited by an "immunogenic amount" of the antigen can be measured by detecting the percent survival or percent death after inoculation of an animal with bacterial cells. In one embodiment, the amount of protection can be measured by measuring at least one symptom associated with bacterial infection, such as fever associated with infection. The amount of each of the antigens in a multi-antigen or multi-component vaccine or immunogenic composition will vary with respect to each of the other components and can be determined by methods known to those skilled in the art. Such methods would include, for example, procedures for measuring immunogenicity and / or in vivo efficacy.

[0303] The term "immunogenic composition" refers to any pharmaceutical composition that contains an antigen, e.g., a microorganism, or a component thereof, and can be used to elicit an immune response in a subject. The immunogenic compositions of the present invention can be used to treat humans susceptible to GBS infections by administering the immunogenic compositions via systemic transdermal or mucosal routes. These administrations can include injection via intramuscular (im), intraperitoneal (ip), intradermal (id) or subcutaneous routes, application by a patch or other transdermal delivery device, or via mucosal administration to the oral / alimentary, respiratory or genitourinary tract. In one embodiment, the immunogenic compositions can be used in the manufacture of vaccines or in the elicitation of polyclonal or monoclonal antibodies that can be used to passively protect or treat animals.

[0304] 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 biological equivalent 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 group B Streptococcus serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX, and wherein the capsular polysaccharide has a sialic acid level of greater than about 60%.In another example, the immunogenic composition comprises a polysaccharide-protein conjugate, wherein the conjugate comprises capsular polysaccharide from group B Streptococcus serotype VI and at least one additional serotype selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII and IX. In another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotype VI and at least two additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII and IX. In yet another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotype VI and at least three additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII and IX. In a further embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotype VI and at least four additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII and IX. In a particular embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotypes VI, VII, VIII and IX, hi another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides from group B Streptococcus serotypes Ia, Ib, II, III and VI.In yet another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate comprising capsular polysaccharides from group B Streptococcus serotype VI and at least five additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII and IX. In one such embodiment, the immunogenic composition comprises six polysaccharide-protein conjugates comprising capsular polysaccharides from group B Streptococcus serotypes Ia, Ib, II, III, IV and VI.

[0305] In one embodiment, the immunogenic composition of the invention comprises one to ten different serotypes of S. agalactiae. Thus, in one embodiment, the immunogenic composition of the invention is a 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-valent GBS conjugate composition. In one such embodiment, the immunogenic composition is a monovalent GBS conjugate composition. In another embodiment, the immunogenic composition is a 2-, 3-, 4-, 5-, or 6-valent GBS conjugate composition. In yet another embodiment, the immunogenic composition is a 7-valent GBS conjugate composition. In a further embodiment, the immunogenic composition is an 8-valent GBS conjugate composition.

[0306] Thus, the present invention relates to monovalent and / or multivalent immunogenic compositions comprising polysaccharide-protein conjugates comprising at least one, two, three or 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. In a specific embodiment, the immunogenic composition comprises GBS capsular polysaccharide serotype VI.

[0307] The polysaccharide-protein conjugates may comprise the same or different protein carriers. In one embodiment, the conjugates comprise the same protein carrier, and the saccharides are conjugated to the same molecule of protein carrier (the carrier molecule having two or more different polysaccharides conjugated thereto). See, for example, International Patent Application Publication No. WO2004 / 083251. In another embodiment, one or more polysaccharides are each independently conjugated to different molecules of protein carrier (each molecule of protein carrier having only one type of polysaccharide conjugated thereto). In such an embodiment, the capsular saccharide is said to be independently conjugated to the carrier protein.

[0308] Optimal amounts of components for a particular immunogenic composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. After an initial vaccination, subjects can receive one or several booster immunizations at appropriately spaced intervals.

[0309] The immunogenic composition of the present invention may further comprise one or more preservatives in addition to the multiple capsular polysaccharide protein conjugates. The FDA requires that biological products in multi-dose (multi-dose) vials contain a preservative, with few exceptions. The present invention contemplates the use of such multi-dose vials. Vaccine products that contain preservatives include vaccines that contain benzethonium chloride (anthrax), 2-phenoxyethanol (DTaP, HepA, Lyme, Polio (parenteral)) and phenol (pneumonia, typhoid (parenteral)). Preservatives approved for use in injectables include, for example, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol and phenylmercuric nitrate.

[0310] In another aspect, the invention relates to a composition including at least one of any of the polysaccharides described herein and a pharma- ceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic surfactant, inhibitor of free radical oxidation, diluent or carrier, or a mixture thereof.

[0311] The immunogenic composition comprises one or more physiologically acceptable buffers selected from, but not limited to, HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. In a preferred embodiment, the buffer is histidine.

[0312] In one embodiment, the immunogenic composition comprises a buffer at a concentration of about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, or about 15 mM to about 20 mM. In a preferred embodiment, the immunogenic composition comprises a buffer at a concentration of about 10 mM to about 25 mM, most preferably about 20 mM.

[0313] In another embodiment, the immunogenic composition comprises histidine at a concentration of about 20 mM.

[0314] In certain embodiments, the formulation is buffered to a pH 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, most preferably about 6.5.

[0315] The immunogenic composition comprises one or more non-ionic surfactants, including but not limited to polyoxyethylene sorbitan fatty acid esters, polysorbate-80 (Tween 80), polysorbate-60 (Tween 60), polysorbate-40 (Tween 40), polysorbate-20 (Tween 20), and polyoxyethylene alkyl ethers, including but not limited to BRIJ® 58, BRIJ® 35, and others, such as Triton X-100, Triton X-114, NP40, Span 85, and the Pluronic® series of non-ionic surfactants (e.g., Pluronic® 121). In one embodiment, the immunogenic composition comprises polysorbate-80 or polysorbate-40, preferably polysorbate-80 (PS80).

[0316] In one embodiment, the immunogenic composition comprises a surfactant at about 0.001% to about 2% (v / w), about 0.001% to about 1%, about 0.001% to about 0.5%, about 0.001% to about 0.1%, about 0.001% to about 0.05%, about 0.001% to about 0.01%, about 0.001% to 0.005%, about 0.005% to about 2%, about 0.005% to about 1%, about 0.005% to about 0.5%, about 0.005% to about 0.1%, about 0.005% to about 0.05%, about 0.005% to about 0.01%, about 0.01% to about 2%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.01% to about 0.1%, about 0.01% to about 0.05%, about 0.01% to about 0.04%, about 0.01% to about 0. .03%, about 0.015% to about 2%, about 0.015% to about 1%, about 0.015% to about 0.5%, about 0.015% to about 0.1%, about 0.015% to about 0.05%, about 0.015% to about 0.04%, about 0.015% to about 0.03%, about 0.02% to about 2%, about 0.02% to about 1%, about 0.02% to about 0.5%, about 0.02% to about 0. In a preferred embodiment, the immunogenic composition comprises a surfactant at a concentration of about 0.01% to 0.03%, about 0.02% to about 0.05%, about 0.02% to about 0.04%, about 0.02% to about 0.03%, about 0.05% to about 2%, about 0.05% to about 1%, about 0.05% to about 0.5%, about 0.05% to about 0.1%, about 0.1% to about 2%, about 0.1% to about 1%, about 0.1% to about 0.5%, or about 0.1% to 0.25%. In a preferred embodiment, the immunogenic composition comprises a surfactant at a concentration of about 0.01% to 0.03%, most preferably about 0.02%.

[0317] In another embodiment, the immunogenic composition comprises polysorbate-80 (PS80) at a concentration of about 0.001% to about 2%, preferably up to about 0.25%, or polysorbate-40 at a concentration of about 0.001% to 1%, preferably up to about 0.5%.

[0318] In one embodiment, the immunogenic composition comprises PS80 at a concentration of about 0.02%.

[0319] A pharmaceutically acceptable carrier should not be confused with a "carrier protein" that is used in binding the carbohydrate of the present invention to a protein and modifies the immune response to the carbohydrate. To avoid confusion with the protein carrier described herein, the term pharmaceutically acceptable diluent is preferred to pharmaceutically acceptable carrier, although these terms may sometimes be used interchangeably. The term "pharmaceutically acceptable carrier" refers to a carrier approved by a federal regulatory agency, a state government or other regulatory agency, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, including humans and non-human mammals. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a 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 may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, and the like. Water, water for injection (WFI), sterile isotonic saline, phosphate buffer solution, adjuvant suspension, aqueous dextrose and glycerol solution, and combinations thereof can be used as liquid carriers, particularly for injectable solutions. Pharmaceutically acceptable diluents may further contain small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, that enhance shelf life or effectiveness in the body. The preparation and use of pharma-ceutically acceptable diluents are well known in the art. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. In one embodiment, the diluent is water, water for injection (WFI), adjuvant suspension, or saline. In certain embodiments, 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.

[0320] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone flour, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. In a preferred embodiment, the excipient is NaCl.

[0321] In one embodiment, the immunogenic composition comprises an excipient at a concentration of about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 10 mM to about 50 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM , about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 150 mM, about 20 mM to about 100 mM, about 20 mM to about 50 mM, about 20 mM to about 30 mM, 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 150 mM to about 500 mM, about 150 mM to about 450 mM, about 150 mM to about 400 mM, about 150 mM to about 350 mM, about 150 mM to about 300 mM, about 150 mM to about 250 mM, about 150 mM to about 200 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 40 0 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 500 mM,In a preferred embodiment, the immunogenic composition comprises an excipient at a concentration of about 10 mM to about 250 mM, most preferably about 150 mM.

[0322] In another embodiment, the excipient is NaCl at a concentration of about 150 mM.

[0323] The compositions can also contain minor amounts of wetting, bulking, emulsifying agents or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, lyophilized powders or cakes, etc. The formulation should be suitable for the mode of administration. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the immunogenic compositions of the present invention is contemplated.

[0324] In one embodiment, the immunogenic composition is lyophilized, optionally in the presence of at least one excipient. In a preferred embodiment, the at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol. In a preferred embodiment, the at least one excipient is selected from the group consisting of sucrose, mannitol and glycine. In a particular embodiment, the at least one excipient is sucrose. In another embodiment, the lyophilized composition comprises an additional excipient. In one such embodiment, the additional excipient is mannitol or glycine.

[0325] In another embodiment, the lyophilized composition comprises at least one sugar at about 1% (w / v) to about 10% (w / v), such as about 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10.0%. In a preferred embodiment, the lyophilized composition comprises at least one excipient at more than about 5.5% (w / v), such as more than about 7.0% (w / v). In further embodiments, the lyophilized composition comprises about 1% (w / v) to about 10% (w / v) of an additional excipient, such as about 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10.0%, etc. In a preferred embodiment, the lyophilized composition comprises about 1% (w / v) to about 10% (w / v) of at least one excipient and about 1% (w / v) to about 10% (w / v) of an additional excipient.

[0326] In yet another embodiment, the lyophilized composition is reconstituted with water, water for injection (WFI), an adjuvant suspension or saline. In a preferred embodiment, the diluent is an aluminum-based adjuvant suspension, such as an aluminum phosphate suspension.

[0327] In one embodiment, the composition comprises the isolated polysaccharide and carrier molecule described herein.Suitable carrier molecules may comprise proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles.Examples of particulate carriers include those derived from polymethylmethacrylate polymers, and microparticles derived from poly(lactide) and poly(lactide-co-glycolide), known as PLG.

[0328] The immunogenic composition of the present invention may further comprise one or more additional "immunomodulators", which are agents that disrupt or modify the immune system, so that either upregulation or downregulation of humoral and / or cell-mediated immunity is observed. In one particular embodiment, upregulation of the humoral and / or cell-mediated arm of the immune system is preferred. Examples of certain immunomodulators include adjuvants or cytokines, such as those described in U.S. Pat. No. 5,254,339, among others, 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.

[0329] Non-limiting examples of adjuvants that can be used in the compositions of the invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.); mineral gels, such as aluminum hydroxide gel; water-in-oil emulsions, such as Freund's complete and incomplete adjuvants; block copolymers (CytRx, Atlanta Ga.); SAF-M (Chiron, Emeryville, Calif.); AMPHIGEN® adjuvant; saponin; Quil A or other saponin fractions; monophosphoryl lipid A; and avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful as adjuvants in the vaccines of the invention include MF59 (U.S. Pat. No. 6,299,884) (containing 5% squalene, 0.5% polysorbate 80, and 0.5% Span 85 (which may contain various amounts of MTP-PE) formulated into submicron particles using a microfluidizer, such as the Model 110Y Microfluidizer (Microfluidics, Newton, Mass.)), and SAF (microfluidized into a submicron emulsion or or vortexed to produce larger particle size emulsions; modified SEAM62 (containing 5% (v / v) squalene (Sigma), 1% (v / v) SPAN® 85 surfactant (ICI surfactant), 0.7% (v / v) polysorbate 80 surfactant (ICI surfactant), 2.5% (v / v) ethanol, 200 μg / ml Quil A, containing 100 μg / ml cholesterol and 0.5% (v / v) lecithin); and modified SEAM 1 / 2, containing 5% (v / v) squalene, 1% (v / v) SPAN® 85 surfactant, 0.7% (v / v) polysorbate 80 surfactant, 2.5% (v / v) ethanol, 100 μg / ml Quil A and 50 μg / ml cholesterol.

[0330] Suitable adjuvants used to enhance immune responses further include, but are not limited to, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT), described in U.S. Patent No. 4,912,094. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGPs), available from Corixa (Hamilton, MT), and described in U.S. Patent No. 6,113,918, or derivatives or analogs thereof. One such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxy-tetrade-canoyl)]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-bD-glucopyranoside, also known as 529 (formerly known as RC529). The 529 adjuvant is formulated as an aqueous form (AF) or as a stable emulsion (SE).

[0331] Still other adjuvants include cyclodextrin derivatives (U.S. Pat. No. 6,165,995); polyanionic polymers (U.S. Pat. No. 6,610,310); muramyl peptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) and N-acetyl-normuramyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE); Amphigen; avridine; L121 / squalene; D-lactide-polylactide / glycoside; Pluronic® polyols; killed Bordetella; saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, Mass.), as described in U.S. Pat. No. 5,057,540; Mycobacterium tuberculosis; tuberculosis); bacterial lipopolysaccharides; synthetic polynucleotides such as oligonucleotides containing CpG motifs (e.g., U.S. Pat. No. 6,207,646); IC-31 (Intercell AG, Vienna, Austria), described in European Patents Nos. 1,296,713 and 1,326,634; pertussis toxin (PT) or mutants thereof, cholera toxin or mutants thereof (e.g., U.S. Pat. Nos. 7,285,281, 7,332,174, 7,361,355 and 7,384,640); or E. coli heat-labile toxin (LT) or mutants thereof, in particular LT-K63, LT-R72 (e.g., U.S. Pat. Nos. 6,149,919, 7,115,730 and 7,291,588).

[0332] Other "immunomodulatory agents" that may be included in the vaccine include, for example, one or more of interleukin 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and mutant forms thereof); interferon-α, β and γ; granulocyte-macrophage colony-stimulating factor (GM-CSF) (see, e.g., U.S. Pat. No. 5,078,996 and ATCC Accession No. 39900); macrophage colony-stimulating factor (M-CSF); granulocyte colony-stimulating factor (G-CSF); or tumor necrosis factors α and β. Still other adjuvants useful in the immunogenic compositions described herein include chemokines, including, but not limited to, MCP-1, MIP-1α, MIP-1β, and RANTES; adhesion molecules, such as selectins, such as L-selectin, P-selectin, and E-selectin; mucin-like molecules, such as CD34, GlyCAM-1, and MadCAM-1; members of the integrin family, such as LFA-1, VLA-1, Mac-1, and p150.95; members of the immunoglobulin superfamily, such as PECAM, ICAMs, such as such as ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3; costimulatory molecules such as B7-1, B7-2, CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2 and DR6; and caspases (ICE).

[0333] It should be understood that the decision to use or not use an immunomodulatory agent and / or adjuvant, or the choice of which immunomodulatory agent and / or adjuvant to use, is expected to depend on the subject to which the vaccine or immunogenic composition is administered, the route of injection, and the number of injections to be given. For example, if the subject has been naturally exposed to a pathogen, an adjuvant may not be required because the vaccine antigen can effectively induce a memory response. In certain embodiments, the immunogenic composition will include one or more adjuvants. In one embodiment, the immunogenic composition comprises streptococcal C5a peptidase (SCP). 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 hydroxyl phosphate. In a particular embodiment, the adjuvant is aluminum phosphate. In another embodiment of the invention, the immunogenic composition comprises QS-21 as an adjuvant.

[0334] In one embodiment, the immunogenic composition comprises an adjuvant at a concentration of about 0.1 mg / ml to about 1.0 mg / ml, 0.1 mg / ml to about 0.9 mg / ml, 0.1 mg / ml to about 0.8 mg / ml, 0.1 mg / ml to about 0.7 mg / ml, 0.1 mg / ml to about 0.6 mg / ml, 0.1 mg / ml to about 0.5 mg / ml, 0.1 mg / ml to about 0.4 mg / ml, 0.1 mg / ml to about 0.3 mg / ml, 0.1 mg / ml to about 0.2 mg / ml, 0.25 mg / ml to about 0.95 mg / ml, 0.25 mg / ml to about 0.85 mg / ml, 0.25 mg / ml to about 0.75 mg / ml, 0.25 mg / ml to about 0.65 mg / ml, 0.25 mg / ml to about 0.5 mg / ml, 0.25 mg / ml to about 0.65 mg / ml, 0.25 mg / ml to about 0.75 mg / ml, 0.25 mg / ml to about 0.8 ... In some embodiments, the present invention is directed to a method for treating or preventing osteoporosis, comprising administering to a subject an effective amount of the present invention at a concentration ranging from about 0.55 mg / ml, 0.25 mg / ml to about 0.45 mg / ml, 0.25 mg / ml to about 0.35 mg / ml, 0.5 mg / ml to about 1.0 mg / ml, 0.5 mg / ml to about 0.9 mg / ml, 0.5 mg / ml to about 0.8 mg / ml, 0.5 mg / ml to about 0.75 mg / ml, 0.5 mg / ml to about 0.7 mg / ml, 0.5 mg / ml to about 0.65 mg / ml, 0.5 mg / ml to about 0.6 mg / ml, 0.75 mg / ml to about 1.0 mg / ml, 0.75 mg / ml to about 0.95 mg / ml, 0.75 mg / ml to about 0.9 mg / ml, and 0.75 mg / ml to about 0.85 mg / ml. In a preferred embodiment, the immunogenic composition comprises an adjuvant at a concentration of about 0.25 mg / ml to about 0.75 mg / ml, most preferably about 0.5 mg / ml.

[0335] In another embodiment, the adjuvant is aluminum-based at a concentration of about 0.5 mg / ml. In one such embodiment, the aluminum-based adjuvant is aluminum phosphate or aluminum hydroxyl phosphate.

[0336] In one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate as described herein, a buffer, a surfactant, an excipient, and optionally an adjuvant, and the composition is buffered to a pH of about 6.0 to about 7.0.

[0337] In one such embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the composition is buffered to a pH of about 6.0 to about 7.0, and the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0338] In one embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the composition is buffered to a pH of about 6.0 to about 7.0, and the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0339] In another embodiment, the immunogenic composition comprises about 5 mcg / ml to about 50 mcg / ml of GBS polysaccharide-protein conjugate, about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0340] In one such embodiment, the immunogenic composition comprises at least one GBS polysaccharide-protein conjugate, a buffer, a surfactant, an excipient, and optionally an adjuvant, and the composition is buffered to a pH of about 6.0 to about 7.

[0341] In one such embodiment, the immunogenic composition comprises at least two GBS polysaccharide-protein conjugates, a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the composition is buffered to a pH of about 6.0 to about 7.0, and the conjugates comprise capsular polysaccharides from Group B Streptococcus (GBS) serotype VI and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, IV, V, VII, VIII and IX.

[0342] In a preferred embodiment, the immunogenic composition comprises about 5 mcg / ml to about 50 mcg / ml each of at least two GBS polysaccharide-protein conjugates, about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate, wherein the conjugates comprise capsular polysaccharides from Group B Streptococcus (GBS) serotype VI and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, IV, V, VII, VIII and IX.

[0343] Evaluation of immunogenic compositions Various in vitro tests are used to assess the immunogenicity of the immunogenic compositions of the present invention. For example, an in vitro opsonization assay is performed by incubating together a mixture of streptococcal cells, heat-inactivated serum containing specific antibodies against the antigen of interest, and an exogenous complement source. Opsonophagocytosis proceeds during incubation of the antibody / complement / streptococcal cell mixture with freshly isolated polymorphonuclear cells (PMN) or differentiated effector cells such as HL60. Antibody and complement coated bacterial cells are killed during opsonophagocytosis. The viable bacterial colony forming units (cfu) recovered from opsonophagocytosis are determined by plating the assay mixture. Titers are reported as the reciprocal of the highest dilution that gives 50% bacterial killing as determined by comparison with an assay control.

[0344] Whole cell ELISA assays may be used to assess in vitro immunogenicity and surface exposure of antigens, where the bacterial strain of interest (S. agalactiae) is coated onto a plate, such as a 96-well plate, and test sera from immunized animals are allowed to react with the bacterial cells. Antibodies specific for the test antigen, if reactive with a surface-exposed epitope of the antigen, can be detected by standard methods known to those of skill in the art. Alternatively, flow cytometry may be used to measure surface exposure of capsular polysaccharide antigens and specificity of antibodies, including monoclonal antibodies.

[0345] Antigens demonstrating the desired in vitro activity may then be tested in an in vivo animal challenge model. In certain embodiments, the immunogenic compositions are used in immunizing animals (e.g., mice) by methods and routes of immunization known to those of skill in the art (e.g., intranasal, parenteral, oral, rectal, intravaginal, transdermal, intraperitoneal, intravenous, subcutaneous, etc.). After immunization of animals with the GBS immunogenic compositions, the animals are challenged with a Streptococcus agalactiae strain and assayed for resistance to streptococcal infection.

[0346] In one embodiment, pathogen-free mice are immunized and inoculated with S. agalactiae. For example, mice are immunized with one or more doses of desired antigen in immunogenic composition. Then, mice are inoculated with S. agalactiae and survival is monitored over time after inoculation.

[0347] How to use "Immunocompromised" as used herein refers to a subject suffering from a deficiency in the cellular and / or humoral arms of the immune system. Thus, degrees of deficiency in immune function ranging from slight impairment in immune processes to complete immunosuppression are contemplated.

[0348] The term "subject" refers to a mammal, bird, fish, reptile, or any other animal. The term "subject" also includes humans. The term "subject" also includes household pets. Non-limiting examples of household pets include dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, birds, snakes, lizards, fish, turtles, and frogs. The term "subject" also includes livestock animals. Non-limiting examples of livestock animals include alpacas, bison, camels, cows, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

[0349] As used herein, "treatment" (including variations thereof, such as "treat" or "treated") refers to any one or more of the following: (i) prevention of infection or reinfection, such as traditional vaccines; (ii) reduction in severity or elimination of symptoms; and (iii) substantial or complete elimination of the pathogen or disorder in question. Thus, treatment can be accomplished prophylactically (before infection) or therapeutically (after infection). Prophylactic or therapeutic treatments can be used in the present invention. According to certain embodiments of the present invention, compositions and methods are provided that include prophylactically and / or therapeutically immunizing and treating a host animal against microbial infections (e.g., bacteria such as S. agalactiae). The methods of the present invention are useful for conferring prophylactic and / or therapeutic immunity to a subject. The methods of the present invention can also be performed in subjects for biomedical research applications.

[0350] In another aspect, the invention relates to a method of inducing an immune response against GBS in a subject by administering to the subject an effective amount of the immunogenic composition described herein. In one embodiment, the invention relates to a method of preventing or reducing a disease or condition associated with group B streptococcus in a subject by administering to the subject an effective amount of the immunogenic composition described herein. In an aspect, the invention relates to an immunogenic composition described herein for use as a medicament. In an aspect, the invention relates to an immunogenic composition described herein for use in a method of inducing an immune response against GBS in a subject. In a particular embodiment, the subject is a woman planning a pregnancy or a pregnant woman. In one such embodiment, the pregnant woman is in the third or second trimester of pregnancy, such as at least 20 weeks or at least 27 weeks of pregnancy. In a preferred embodiment, the pregnant woman is between 27 and 36 weeks of pregnancy. In another embodiment, the subject is an elderly person, such as an adult aged 50 years or older, 65 years or older, and 85 years or older. In a further embodiment, the subject is immunocompromised. In one aspect, the subject may have a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease. In a preferred embodiment, the group B streptococcus is S. agalactiae.

[0351] The immunogenicity or effective amount of the immunogenic composition can be determined by performing a dose-response study in which subjects are immunized with increasing amounts of the immunogenic composition and the immune response is analyzed to determine the optimal dosage. The starting point of the study can be extrapolated from immunization data in animal models. The dosage can vary depending on the specific condition of the individual. The amount can be determined by routine testing using means known to those skilled in the art.

[0352] The immunogenic composition is administered to the subject in an immunologically effective amount of an appropriate dose to induce an immune response. The dosage may vary according to the specific conditions of the individual, such as age and weight. This amount can be determined by routine testing using means known to those skilled in the art.

[0353] In one embodiment, patients administered the immunogenic composition of the invention show a reduction in S. agalactiae carriage rates. Such a reduction in carriage or an extension of the time interval spent as a non-carrier after administration of the immunogenic composition is significant in terms of medical need. For example, a reduction in overall S. agalactiae carriage in carriers can be assessed after one dose of the immunogenic composition of the invention. For example, a group of adults aged 18-50 years can be screened for carriage by nasal, throat, axillary, rectal, perineal and vaginal swabs one day prior to administration of the immunogenic composition, followed by culture to determine carriage status. The group can then be administered the immunogenic composition of the invention, and one group can receive a control. After administration of the immunogenic composition, nasal, throat, axillary, rectal, perineal and vaginal swabs are performed weekly for 12 weeks and monthly for up to 6 months and compared to a placebo. One primary endpoint will be to compare carriage rates in patients following administration of the immunogenic composition with placebo at three-month intervals following immunization.

[0354] antibody An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless the context indicates otherwise, the term is intended to encompass intact polyclonal or monoclonal antibodies, as well as engineered antibodies (e.g., chimeric, humanized and / or derivatized to modify effector functions, stability and other biological activities) and fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chain (ScFv) and domain antibodies, including shark and camel antibodies), and fusion proteins comprising antibody portions, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity) and antibody fragments as described herein, as well as any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site. Antibodies encompass antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and do not have to be of any particular class. Depending on the amino acid sequence of the antibody in the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 in humans. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0355] An "antibody fragment" comprises only a portion of an intact antibody, which portion preferably retains at least one, and preferably most or all, of the functions normally associated with that portion when present in the intact antibody.

[0356] The "functional activity" of an antibody or a "functional antibody" as used herein refers to an antibody that can at a minimum specifically bind to an antigen. Additional functions are known in the art and may include additional components of the immune system that achieve pathogen clearance or killing, such as through opsonization, ADCC, or complement-mediated cytotoxicity. After antigen binding, any subsequent antibody function may be mediated through the Fc region of the antibody. The antibody opsonophagocytosis assay (OPA) is an in vitro assay designed to measure in vitro Ig complement-assisted killing of bacteria by effector cells (white blood cells), thus mimicking a biological process. Antibody binding may also directly inhibit the biological function of the antigen to which it is bound. In some embodiments, a "functional antibody" refers to an antibody that is functional as measured by bacterial killing in animal efficacy models or opsonophagocytic killing assays that demonstrate that the antibody kills bacteria.

[0357] In one aspect, the invention relates to an isolated antibody or fragment thereof that specifically binds to the polysaccharide described herein. An "isolated" antibody, as used herein, refers to an antibody that has been identified, separated and / or recovered from components of its natural environment. Contaminating components of its natural environment are materials that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In exemplary embodiments, the antibody will be purified (1) to 95% by weight of the antibody, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the antibody's natural environment is expected to be absent. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0358] An antibody that "specifically binds to" or is "specific for" a particular polysaccharide or an epitope on a particular polysaccharide is one that binds to the particular polysaccharide or an epitope on the particular polysaccharide without specifically binding to any other polysaccharide or polysaccharide epitope.

[0359] The term "label," as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. The label may be itself detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0360] The present invention further provides antibodies and antibody compositions that specifically and selectively bind to one or more antigens of the immunogenic composition of the present invention. In some embodiments, the antibodies are produced upon administration of the immunogenic composition of the present invention to a subject. In some embodiments, the present invention provides purified or isolated antibodies directed to one or more of the antigens of the immunogenic composition of the present invention. In some embodiments, the antibodies of the present invention are functional as measured by killing bacteria in any animal efficacy model or via opsonophagocytic killing assay. In some embodiments, the antibodies of the present invention confer passive immunity to a subject. The present invention further provides polynucleotide molecules encoding the antibodies or antibody fragments of the present invention, as well as cells or cell lines (such as hybridoma cells or other engineered cell lines for recombinant production of antibodies) and transgenic animals that produce the antibodies or antibody compositions of the present invention using techniques well known to those skilled in the art.

[0361] The antibodies or antibody compositions of the invention may be used in a method of treating or preventing a streptococcal infection, disease or condition associated with S. agalactiae in a subject, comprising producing a polyclonal or monoclonal antibody preparation and using said antibody or antibody composition to confer passive immunity to the subject. The antibodies of the invention may also be useful in diagnostic methods, e.g., for detecting the presence or quantifying the level of one or more antigens of the immunogenic compositions of the invention.

[0362] The antibody response to repeating structures such as the polysaccharides of the present invention may exhibit some unique features. For example, the regularity of the repeating units may mean that antigen molecules of very different molecular weights can bind to antibodies specific for the polysaccharide. The second repeating structure of the longer polysaccharide can induce a T cell-independent antibody response. Thus, when using polysaccharides conjugated to protein carriers with T cell helper epitopes, both T cell-independent and T cell-dependent antibody responses can be stimulated. Thus, the immune response can be modified by the appropriate selection of polysaccharide size, regardless of whether a carrier protein is used or not.

[0363] Polyclonal antibodies In certain embodiments, the anti-polysaccharide antibody is a polyclonal antibody.Polyclonal antibody as defined herein refers to a mixture of antibodies with different specificities that are derived from serum preparations and originate from different B cell clones.The preparation and characterization of polyclonal antibodies are known in the art.

[0364] Polyclonal antibodies are raised in a subject, e.g., a mammal, by administering one or more injections of the immunogen or immunogenic composition described herein, as well as adjuvants, buffers and / or diluents, if desired. A range of animal species may be useful for the production of specific antisera. Typically, animals used for the production of anti-saccharide polyclonal antisera are non-human primates, goats, sheep, rabbits, mice, rats, hamsters or guinea pigs. Typically, the immunogen or immunogenic composition is injected into the mammal by multiple injections, with or without an adjuvant. The immunogenic material may include polysaccharides, oligosaccharides, polysaccharides, polysaccharide-protein conjugates described herein, or larger aggregates of immunogens. Typically, blood is collected from the immunized animal for the first 2-6 weeks after the initial immunization, allowed to clot, and serum is collected. The serum contains anti-saccharide polyclonal antibodies from the immunized animal and is often referred to as antiserum.

[0365] Monoclonal antibodies Anti-saccharide monoclonal antibodies can be prepared through the use of known hybridoma technology. Typically, preparing monoclonal antibodies involves first immunizing a suitable target animal host with a selected immunogen, including a polysaccharide, oligosaccharide, polysaccharide, or polysaccharide-protein conjugate of the present invention. If desired, adjuvants, buffers, and / or diluents may be included. Immunization is performed in a manner sufficient to induce B lymphocytes to produce or express antibodies that specifically bind to the polysaccharide or its conjugate. Alternatively, lymphocytes are immunized in vitro.

[0366] The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. The origin of the lymphocytes determines whether the monoclonal antibodies are of human or animal origin. Generally, peripheral blood lymphocytes ("PBL") are used when antibodies and cells of human origin are desired, and spleen cells or lymph node cells are used when non-human mammalian sources are desired.

[0367] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. Hybridoma cells are cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will typically contain hypoxanthine, aminopterin and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0368] The immortalized cell line is selected based on practical considerations such as species of origin, fusion and growth characteristics. For example, a suitable immortalized cell line is one that fuses efficiently, supports stable high level expression of antibody by the selected antibody-producing cells, and is sensitive to a medium such as HAT medium. Examples of immortalized cell lines include murine myeloma lines. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

[0369] The monoclonal antibodies are secreted into the culture medium by the hybridoma cells. The culture medium is then assayed for the presence of monoclonal antibodies that recognize and bind to polysaccharides. The anti-polysaccharide binding specificity of a particular monoclonal antibody produced by the hybridoma cells is determined by one of a number of procedures well known to those skilled in the art. For example, the antibody binding specificity can be determined by immunoprecipitation, radioimmunoassay (RIA), Western blot, enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (e.g., Biacore). The exact epitope recognized by the monoclonal antibody is determined by epitope mapping. Such techniques and assays are well known in the art.

[0370] After hybridoma cells producing antibodies with the desired specificity are identified, the clones are subcloned by limiting dilution and cultured using standard methods. Culture media suitable for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells are grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones are isolated and purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as, for example, protein A Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0371] Alternatively, antibodies from a desired species source with the desired specificity can be obtained through the use of phage display libraries. In addition, examples of methods and reagents that are particularly suitable for use in producing and screening antibody display libraries can be found in the art.

[0372] Use of antibodies In one aspect, the present invention relates to the use of the immunogenic compositions described herein to generate GBS antibodies and / or antibody fragments. The polysaccharide-protein conjugates described herein and / or antibodies produced therefrom can be used in various immunodiagnostic techniques known to those skilled in the art, including ELISA and microarray-related techniques. In addition, these reagents can be used to evaluate antibody responses, including serum antibody levels, against the immunogenic polysaccharide conjugates. The assay methodology of the present invention can involve the use of labels, such as fluorescent, chemiluminescent, radioactive, enzyme-labeled or dye molecules, and / or secondary immunological reagents for direct or indirect detection of the complex between the antigen or antibody in the biological sample and the corresponding antibody or antigen bound to a solid support.

[0373] The antibodies or antibody fragments produced may also be useful in passive immunotherapy or for prophylaxis against streptococcal infections.

[0374] Methods for Producing Polysaccharides In yet another aspect, the invention relates to a method for producing the polysaccharides described herein, comprising culturing GBS and harvesting the polysaccharides produced by the bacteria. In one embodiment, the GBS comprises S. agalactiae. The bacteria may be any strain of S. agalactiae. In a preferred embodiment, the bacteria is an encapsulated strain of S. agalactiae. S. agalactiae strains for use in the present invention include 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524, MB4052 (ATCC Accession No. 31574), H36B (ATCC Accession No. 12401), S40, S42, MB4053 (ATCC Accession No. 31575), M709, 133, 7 357, PFEGBST0267, MB4055 (ATCC accession no. 31576), 18RS21 (ATCC accession no. BAA-1175), S16, S20, V8 (ATCC accession no. 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB4082 (ATCC accession no. 31577), M132, 110, M781 (ATCC accession no. BAA-22), D136C(3)(AT ATCC accession no. 12403), M782, S23, 120, MB4316 (M-732; ATCC accession no. 31475), M132, K79, COH1 (ATCC accession no. BAA-1176), PFEGBST0563, 3139 (ATCC accession no. 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession no. BAA-23), CJB112, and 2603 V / R (ATCC Accession No. BAA-611), NCTC10 / 81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B4589, B4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62, and IT-PW-64.

[0375] The polysaccharides described herein can be produced by culturing GBS in a suitable medium. The suitable medium can include Columbia broth. The medium can include dextrose, hemin and / or glucose. In an embodiment, the medium includes Columbia broth and dextrose. When S. agalactiae is cultured using Columbia broth and dextrose, preferably the temperature for culturing is 20 to 40°C, preferably 37°C. In a preferred embodiment, the bacteria is cultured under aerobic conditions. In another preferred embodiment, the bacteria is cultured for 12 to 60 hours.

[0376] The polysaccharides may be collected from the obtained culture by using methods known in the art for collecting target substances from cultures, such as heating, enzyme treatment, centrifugation, precipitation, treatment with activated charcoal, and / or filtration (see, e.g., U.S. Patent Application Publication Nos. 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498; International Patent Application Publication No. WO2008 / 118752). In one embodiment, the culture containing the bacteria and polysaccharides is centrifuged and treated with enzymes, such as lysozyme, RNase, DNase, pronase, mutanolysin, and mixtures thereof. For example, in one embodiment, a suitable organic solvent is added to the obtained supernatant to precipitate proteins, and the precipitate is removed by centrifugation. The polysaccharides may then be precipitated by further adding a suitable organic solvent to the supernatant, and the polysaccharides may be collected by centrifugation. More specifically, the polysaccharides described herein may be obtained by adding ethanol to the bacteria-removed supernatant at a final concentration of about 25% by volume, removing the protein-containing precipitate by centrifugation, further adding ethanol thereto to a final concentration of about 75% by volume, and then collecting the precipitate by centrifugation. The resulting precipitate may be dried with nitrogen. The resulting precipitate may be resuspended in Tris and 0.05% Na azide.

[0377] A further aspect of the present invention provides a novel method using organic reagents such as derivatized hydroxylamine compounds for the isolation of nearly intact high molecular weight CPs while preserving N- and O-acetyl groups. Because this method does not lyse cells, CPs isolated by centrifugation are minimally contaminated by intracellular components, leading to higher overall yields. Moreover, these reagents cleave the B antigen impurity through its multiple phosphodiester bonds into very small fragments that can be easily removed by hemodiafiltration.

[0378] In one embodiment, the CP is isolated by reacting hydroxylamine with a cell paste containing the capsular polysaccharide-producing bacteria. In one embodiment, the method further comprises a centrifugation step. In another embodiment, the method further comprises a filtration step.

[0379] In certain aspects of the invention, the hydroxylamine may be one listed in Table 2 of Example 2. In preferred embodiments, the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine, diethylhydroxylamine, hydroxylamine, ethylenediamine, triethylenetetramine, 1,1,4,7,10,10 hexamethyltriethylenetetramine, and 2,6,10,trimethyl 2,6,10 triazaundecane.

[0380] In one embodiment of the invention, the concentration of hydroxylamine is from about 5 mM to about 200 mM, e.g., from about 5 mM to about 150 mM, from about 5 mM to about 100 mM, from about 5 mM to about 75 mM, from about 5 mM to about 50 mM, from about 5 mM to about 25 mM, from about 5 mM to about 10 mM, 10 mM to about 200 mM, e.g., from about 10 mM to about 150 mM, from about 10 mM to about 100 mM, from about 1 Such as 0 mM to about 75 mM, about 10 mM to about 50 mM, about 10 mM to about 25 mM, about 25 mM to about 200 mM, about 25 mM to about 150 mM, about 25 mM to about 100 mM, about 25 mM to about 75 mM, about 25 mM to about 50 mM, about 50 mM to about 200 mM, about 50 mM to about 150 mM, 50 mM to about 100 mM, and about 50 mM to about 75 mM.

[0381] In another embodiment, the pH of the reaction is maintained at about 5.5 to about 9.5, e.g., about 5.5 to about 9.0, about 5.5 to about 8.5, about 5.5 to about 8.0, about 5.5 to about 7.5, about 5.5 to about 7.0, about 5.5 to about 6.5, about 6.0 to about 9.5, about 6.0 to about 9.0, about 6.0 to about 8.5, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.0 to about 7.0, about 6.5 to about 9.5, about 6.5 to about 8.5, about 6.5 to about 8.0, about 6.5 to about 7.5, about 7.0 to about 9.5, about 7.0 to about 9.0, 7.0 to about 8.5 and about 7.0 to about 8.0.

[0382] In a further embodiment of the invention, the reaction is carried out at a temperature between about 20° C. and about 85° C., e.g., between about 20° C. and about 80° C., between about 20° C. and about 75° C., between about 20° C. and about 70° C., between about 20° C. and about 65° C., between about 20° C. and about 60° C., between about 20° C. and about 55° C., between about 20° C. and about 50° C., between about 25° C. and about 85° C., between about 25° C. and about 80° C., between about 25° C. and about 75° C., between about 25° C. and about 55° C., between about 25° C. and about 50° C., to about 70°C, about 25°C to about 65°C, about 25°C to about 60°C, about 25°C to about 55°C, about 25°C to about 50°C, about 30°C to about 85°C, about 30°C to about 80°C, about 30°C to about 75°C, about 30°C to about 70°C, about 30°C to about 65°C, about 30°C to about 60°C, about 30°C to about 55°C, about 30°C to about 50°C, about 35°C to about 85°C , about 35°C to about 80°C, about 35°C to about 75°C, about 35°C to about 70°C, about 35°C to about 65°C, about 35°C to about 60°C, about 35°C to about 55°C, about 40°C to about 85°C, about 40°C to about 80°C, about 40°C to about 75°C, about 40°C to about 70°C, about 40°C to about 65°C, about 40°C to about 60°C, about 45°C to about 85°C, about 45°C from about 80°C, from about 45°C to about 75°C, from about 45°C to about 70°C, from about 45°C to about 65°C, from about 50°C to about 85°C, from about 50°C to about 80°C, from about 50°C to about 75°C, from about 50°C to about 70°C, from about 55°C to about 85°C, from about 55°C to about 80°C, from about 55°C to about 75°C, from about 60°C to about 85°C, and from about 65°C to about 85°C.

[0383] In yet another embodiment, the reaction time is from about 10 hours to about 90 hours, e.g., from about 10 hours to about 85 hours, from about 10 hours to about 80 hours, from about 10 hours to about 75 hours, from about 10 hours to about 70 hours, from about 10 hours to about 60 hours, from about 10 hours to about 50 hours, from about 10 hours to about 40 hours, from about 10 hours to about 30 hours, from about 10 hours to about 25 hours, from about 10 hours to about 20 hours, from about 10 hours to about 15 hours, from about 15 hours to about 90 hours, from about 15 hours to about 85 hours, from about 15 hours to about 80 hours, from about 15 hours to about 20 hours, about 75 hours, about 15 hours to about 70 hours, about 15 hours to about 60 hours, about 15 hours to about 50 hours, about 15 hours to about 40 hours, about 15 hours to about 30 hours, 15 hours to about 20 hours, such as about 20 hours to about 90 hours, about 20 hours to about 85 hours, about 20 hours to about 80 hours, about 20 hours to about 75 hours, about 20 hours to about 70 hours, about 20 hours to about 60 hours, about 20 hours to about 50 hours, about 20 hours to about 40 hours, about 20 hours to about 30 hours, and about 20 hours to about 25 hours.

[0384] Alternatively, in another embodiment of the invention, the polysaccharides are chemically synthesized. The polysaccharides may be chemically synthesized according to conventional methods.

[0385] In yet another embodiment of the invention, the polysaccharide is prepared by cloning and expressing a biosynthetic pathway for producing the polysaccharide, followed by expression in a surrogate host. For example, the host cell may be modified to produce a polysaccharide having structural similarity to the polysaccharides described herein, where the repeating units of the polysaccharides produced in the host cell are partially identical to the repeating units of the polysaccharides described herein. The polysaccharide is structurally similar to the polysaccharides described herein, e.g., if the repeating units of the polysaccharide have unknown branching, they are non-uniform in size and / or branching arrangement when compared to the repeating units of the polysaccharides described herein. Preferably, the host cell is a bacterial host cell. EXAMPLES

[0386] The following examples demonstrate some embodiments of the present invention. However, it should be understood that these examples are for illustration only and do not claim to be completely definitive on the conditions and scope of the present invention. When typical reaction conditions (e.g., temperature, reaction time, etc.) are given, it should be understood that conditions both above and below the specified ranges can also be used, although generally less favorable. All parts and percentages referred to herein are by weight, and all temperatures are expressed in degrees Celsius unless otherwise specified.

[0387] Additionally, the following examples were performed using standard techniques that are well known and routine to those skilled in the art, unless otherwise specifically described. As noted above, the following examples are presented for illustrative purposes and should not be construed as limiting the scope of the invention in any way.

[0388] Example 1 Preparation of polysaccharide-protein conjugates using O-deacetylated polysaccharides S. agalactiae strains for each serotype were fermented in submerged culture with pH control in defined media. The procedure and media used were optimized through experimentation and were an extension of the basic technique previously described in von Hunolstein, C. et al., Appl. Micro. Biotech. 38(4):458-462 (1993). Capsular polysaccharide was removed from the cells by NaOH treatment. After clarification, a series of UF / DF, precipitation and carbon filtration steps yielded the purified polysaccharide. See, e.g., U.S. Patent No. 8,652,480. Reductive amination chemistry was used to convert the activated polysaccharide to CRM. 197 See, e.g., U.S. Patent No. 5,360,897.

[0389] Example 2 Conjugation of GBS capsular polysaccharide by reductive amination Activating polysaccharides Polysaccharide oxidation was carried out in 100 mM potassium phosphate buffer (pH 6.0±0.5) by sequential addition of calculated amounts of 500 mM potassium phosphate buffer (pH 6.0) and water for injection (WFI) to obtain a final polysaccharide concentration of 2.0 g / L. If necessary, the reaction pH was adjusted to approximately pH 6.0. After pH adjustment, the reaction temperature was adjusted to 23° C. The oxidation was initiated by the addition of approximately 0.25 molar equivalents of sodium periodate. The oxidation reaction was carried out at 5±3° C. for approximately 16 hours.

[0390] Concentration and hemodiafiltration of the activated polysaccharides were performed using 5K MWCO ultrafiltration cassettes. Hemodiafiltration was performed against 20 diavolumes of WFI. The purified activated polysaccharides were then stored at 5±3° C. The purified activated sugars were characterized by, among others, (i) sugar concentration by colorimetric assay, (ii) aldehyde concentration by colorimetric assay, (iii) degree of oxidation, and (iv) molecular weight by SEC-MALLS.

[0391] The degree of oxidation (DO = moles of sugar repeat units / moles of aldehyde) of the activated polysaccharides was determined as follows:

[0392] The number of moles of sugar repeating units is determined by various colorimetric methods, for example, by using the anthrone method. With the anthrone method, polysaccharides are first broken down into monosaccharides by the action of sulfuric acid and heat. The anthrone reagent reacts with hexoses to form a yellow-green complex whose absorbance is read spectrophotometrically at 625 nm. Within the range of the assay, the absorbance is directly proportional to the amount of hexose present.

[0393] The moles of aldehyde are also determined simultaneously using the MBTH colorimetric method. The MBTH assay involves the formation of an azine compound by reacting aldehyde groups (from a given sample) with 3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). Excess 3-methyl-2-benzothiazolone hydrazone is oxidized to form a reactive cation. The reactive cation and azine react to form a blue chromophore. The formed chromophore is then read spectroscopically at 650 nm.

[0394] The activated polysaccharide is combined with sucrose excipient and lyophilized. The activated polysaccharide was blended with sucrose to a ratio of 25 grams of sucrose per gram of activated polysaccharide. The bottles of the blended mixture were then freeze-dried. After freeze-drying, the bottles containing the freeze-dried activated polysaccharide were stored at -20±5°C. The calculated amount of CRM 197 The proteins were shell frozen and lyophilized separately. Lyophilized CRM 197 were stored at -20±5°C.

[0395] Reconstitute the lyophilized activated polysaccharide and carrier protein The lyophilized activated polysaccharide was redissolved in anhydrous dimethyl sulfoxide (DMSO). Once the polysaccharide was completely dissolved, an equal volume of anhydrous DMSO was added to the lyophilized CRM 197 was added to redissolve.

[0396] Conjugate and cap The redissolved activated polysaccharide was added to the redissolved CRM 197and were combined in a reaction vessel and then mixed thoroughly to obtain a clear solution before initiating the conjugation with sodium cyanoborohydride. The final polysaccharide concentration in the reaction solution was approximately 1 g / L. The conjugation was initiated by adding 1.0-1.5 MEq of sodium cyanoborohydride to the reaction mixture and incubating at 23 ± 2 °C for 20-48 h. The conjugation reaction was terminated by adding 2 MEq of sodium borohydride (NaBH4) to cap the unreacted aldehydes. The capping reaction was allowed to continue for 3 ± 1 h at 23 ± 2 °C.

[0397] Purifying the conjugate The conjugate solution was diluted 1:10 with chilled 5 mM succinate-0.9% saline (pH 6.0) in preparation for purification by tangential flow filtration using 100-300K MWCO membranes.

[0398] The diluted conjugate solution was passed through a 5 μm filter and hemodiafiltration was performed using 5 mM succinate / 0.9% saline (pH 6.0) as the medium. After hemodiafiltration was completed, the conjugate concentrate was transferred through a 0.22 μm filter. The conjugate was further diluted with 5 mM succinate / 0.9% saline (pH 6) to a target sugar concentration of approximately 0.5 mg / mL. Alternatively, the conjugate was purified by tangential flow filtration using 100-300K MWCO membranes with 20 mM histidine-0.9% saline (pH 6.5). A final 0.22 μm filtration step was completed to obtain the immunogenic conjugate.

[0399] Example 3 Conjugation of GBS capsular polysaccharide with CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) GBS polysaccharide was dialyzed against WFI to remove buffer salts and lyophilized. Lyophilized polysaccharide (100 mg) was dissolved in WFI (4-5 mg / mL) and activated with CDAP (100 mg; 100 mg / mL in acetonitrile:water, 9:1) for approximately 30 seconds. After activation, 0.2 M triethylamine (4 mL) was added and stirred for approximately 2.5 minutes, followed by the addition of tetanus toxoid (TT) (150 mg, 3 mg / mL in saline). The pH of the reaction was adjusted to 8.8 and stirred at room temperature for 24 hours. The conjugation reaction was quenched with 2 M glycine and the conjugate was passed through a 5 μm filter and purified using saline by tangential flow filtration using a 100K MWCO membrane. A final 0.22 μm filtration step was completed to obtain the immunogenic conjugate.

[0400] Example 4 GBS polysaccharide-CRM 197 Effect of different conjugation conditions on the conjugates GBS serotype VI, VII, VIII and IX conjugates can be produced by deliberately varying periodate oxidation / reductive amination chemistry (PO / RAC) conditions, including the solvent for the reagents (aqueous media vs. DMSO), different levels of sialic acid in the initial polysaccharide, and the degree of oxidation / glyco-epitope modification. In general, conjugates produced using DMSO as the solvent are found to have lower levels of unreacted (free) polysaccharide, higher conjugate molecular weight and higher sugar / protein ratio than conjugates produced using aqueous media.

[0401] Conjugation processes that produce conjugates with low levels of unreacted (free) polysaccharide are advantageous and preferred, as it is well known that high levels of unreacted (free) polysaccharide can lead to excessive T-cell-independent immune responses that have the potential to dilute the T-cell-dependent response produced by the polysaccharide-protein conjugate, thereby reducing the immunogenic response produced by the conjugate.

[0402] GBS polysaccharides can be chemically desialylated by methods known in the art (see Chaffin, DO et al., J Bacteriol 187(13):4615-4626 (2005)) to produce conjugate mutants to determine the effect of % desialylation on immunogenicity. In general, greater than about 40% desialylation (i.e., sialic acid levels less than about 60%) has a detrimental effect on immunogenicity.

[0403] Similarly, in most cases, a degree of oxidation below about 5 or more than about 20% glycoepitope modification has a detrimental effect on immunogenicity. Because oxidation occurs via sialic acid on the capsular polysaccharide, it is understood that more than about 20% glycoepitope modification reduces the sialic acid content, which results in reduced immunogenicity.

[0404] Conversely, conjugates with a variety of sugar / protein ratios or polysaccharide molecular weights have been shown to generate immunogenic responses in mice, indicating a relatively wide range of acceptance criteria with respect to these attributes.

[0405] Additional conjugate variants can also be produced using alternative chemical routes. One alternative chemistry involves producing conjugates by reacting polysaccharides with carbonylditriazole (CDT) and carrying out the conjugation reaction in DMSO. In another alternative chemistry, conjugates can be produced by oxidation of polysaccharides using TEMPO [(2,2,6,6-tetramethylpiperidin-1-yl)oxyl] reagent (instead of sodium periodate) followed by conjugation using reductive amination chemistry (TEMPO / RAC) in DMSO, as detailed in the examples above. Conjugates produced by these alternative chemistries have been found to be immunogenic in mice, indicating the suitability of alternative chemical routes other than PO / RAC. However, some conjugation chemistries may perform better with some serotypes than others.

[0406] OPA was performed as in Buurman, E. T. et al., J. Infect. Dis., Jun 5;220(1):105-115 (2019).

[0407] Example 5 Monovalent conjugate vaccines generated functional and binding antibody responses in rabbits Rabbits (4-5 per group) were vaccinated at weeks 0, 3, and 6 with 20 mcg by weight of polysaccharide of monovalent conjugates of GBS CPS serotypes VI, VII, VIII, or IX conjugated to different carrier proteins (CRM197, tetanus toxoid (TT), SCP) formulated with 20 mcg of QS21 per conjugate dose. Sera were assessed for anti-CPS IgG titers at baseline and week 10 by direct binding Luminex immunoassay (dLIA) using CPS-coated microspheres.

[0408] Figure 1 shows the average binding activity profile of serially diluted sera from two rabbits administered GBS CPS serotype VI-CRM197 conjugate. The interpolated EC50 serum dilution titers for GBS CPS VI shown in Figure 1 were determined using sigmoidal dose-response curve fitting (variable slope) (GraphPad Prism). Table 1 shows the antibody titers produced for GBS CPS serotype VI conjugated with CRM197, SCP and TT. Immunization with GBS CPS serotype VI conjugated with CRM197 produced measurable antibody titers specific for serotype VI, and negligible binding to other serotypes was seen for serotype VI-elicited sera as assessed by direct binding Luminex immunoassay.

[0409] [Table 1]

[0410] As shown in Figures 2A-2C and Table 2, conjugates of GBS CPS serotypes VII, VIII, or IX conjugated with TT similarly elicited robust IgG antibody titers against the cognate polysaccharide following immunization.

[0411] [Table 2]

[0412] Figures 3A and 3B show the average avidity profiles of serially diluted sera from two rabbits administered GBS CPS VII-TT conjugate and GBS CPS IX-TT conjugate, respectively. Cross-reactivity was observed for GBS CPS VII-TT conjugate-induced antibodies against GBS CPS serotype V and IX antigens, and for GBS CPS IX-TT conjugate-induced antibodies against GBS CPS VI and VII antigens. The interpolated EC50 serum dilution titers shown in Figures 3A (GBS CPS V, VII and IX) and 3B (GBS CPS VI, VII and IX) were determined using sigmoidal dose-response curve fitting (variable slope) (GraphPad Prism).

[0413] GBS CPS serotype VI conjugates with CRM197 or C5a peptidase (SCP) as carrier proteins were further evaluated for eliciting functional bactericidal antibodies capable of killing by opsonophagocytic uptake (OPA). Rabbits were immunized with GBS CPS VI conjugated to CRM197 at weeks 0, 3, and 6, or with CPS VI conjugated to SCP at weeks 0, 3, 6, and 13. Sera from rabbits immunized with GBS VI-CRM197 or GBS VI-SCP, collected at weeks 13 and 16, respectively, were evaluated for OPA titers with HL-60 phagocytic cells (Table 3).

[0414] [Table 3]

[0415] Sera from rabbits immunized with GBS VI-CRM197 or VI:SCP demonstrated killing activity by OPA with target strains expressing serotype VI capsule. Preadsorption of GBS VI-SCP-elicited sera with soluble SCP protein before OPA analysis slightly reduced the observed titers, demonstrating specificity. Negligible cross-reactive killing by GBS VI-CRM197-elicited sera was seen with target strains expressing heterologous serotypes III, VIII, or IX. In comparison, sera from serotype GBS VI-SCP-immunized rabbits demonstrated measurable titers against strains expressing heterologous serotypes III, VIII, or IX. Adsorption of SCP-specific antibodies with soluble SCP effectively reduced the observed titers, supporting the ability of anti-SCP antibodies to achieve functional activity across isolates in the context of diverse capsular types.

[0416] These results indicate that functional activity by anti-CPS antibodies is largely restricted to the homologous serotype and that anti-SCP carrier antibodies can confer a moderate but broad level of CPS-independent cross-functional activity among diverse isolates expressing different CPS types.

[0417] Example 6 Multivalent conjugate vaccines generated combined antibody responses in rabbits A single rabbit was vaccinated with CRM197 conjugates of serotypes Ia, Ib, II, III, IV, V and VI using a 20mcg dose of each antigen and administered 20mcg of QS21 adjuvant at weeks 0, 3, 6 and 9. IgG titers in serum were assessed at week 11 using the Luminex assay described in Example 5. Three-fold serial dilutions of serum were tested starting at 1:15.

[0418] Figure 4 shows serologic responses for the seven administered serotypes Ia, Ib, II, III, IV, V and VI-CRM197 conjugates (open symbols) and the three formulation-derived absent serotypes (VII, VIII, IX) (closed symbols). The results demonstrate that the heptavalent GBS glycoconjugate vaccine encompassing the newly emerged serotype VI can elicit antigen-specific IgG antibodies against all seven capsular polysaccharide components. The responses against serotype VII and IX polysaccharides may be due to cross-reactivity of antibodies against serotype V antigen, which share a common epitope within the branched sialylated side chain of the repeating unit (Berti, F. et al., (2014) JBC 289:34 23437-23448).

[0419] Aspects of the invention The following clauses describe additional embodiments of the present invention.

[0420] C1. An immunogenic polysaccharide-protein conjugate comprising a Group B Streptococcus (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%.

[0421] C2. An immunogenic conjugate of C1, wherein the capsular polysaccharide is selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

[0422] C3. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype VI.

[0423] C4. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype VII.

[0424] C5. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype VIII.

[0425] C6. An immunogenic conjugate of C2 in which the capsular polysaccharide is serotype IX.

[0426] C7. The immunogenic conjugate of any one of C1-C6, wherein the capsular polysaccharide has a sialic acid level of greater than about 95%.

[0427] C8. The immunogenic conjugate of any one of C1-C7, wherein the capsular polysaccharide has a sialic acid level of about 100%.

[0428] C9. The immunogenic conjugate of any one of C1-C8, wherein the capsular polysaccharide has at least about 0.6 mM sialic acid per mM polysaccharide.

[0429] C10. The immunogenic conjugate of any one of C1-C9, wherein the capsular polysaccharide has at least about 0.65 mM sialic acid per mM polysaccharide.

[0430] C11. The immunogenic conjugate of any one of C1-C10, wherein the capsular polysaccharide has at least about 0.7 mM sialic acid per mM polysaccharide.

[0431] C12. The immunogenic conjugate of any one of C1-C11, wherein the capsular polysaccharide has at least about 0.75 mM sialic acid per mM polysaccharide.

[0432] C13. The immunogenic conjugate of any one of C1-C12, wherein the capsular polysaccharide has at least about 0.8 mM sialic acid per mM polysaccharide.

[0433] C14. The immunogenic conjugate of any one of C1-C13, wherein the capsular polysaccharide has at least about 0.85 mM sialic acid per mM polysaccharide.

[0434] C15. The immunogenic conjugate of any one of C1-C14, wherein the capsular polysaccharide has at least about 0.9 mM sialic acid per mM polysaccharide.

[0435] C16. The immunogenic conjugate of any one of C1-C15, wherein the capsular polysaccharide has at least about 0.95 mM sialic acid per mM polysaccharide.

[0436] C17. The immunogenic conjugate of any one of C1-C16, wherein the capsular polysaccharide has a molecular weight between about 5 kDa and about 1,000 kDa.

[0437] C18. The immunogenic conjugate of any one of C1-C17, wherein the capsular polysaccharide has a molecular weight of between about 25 kDa and about 750 kDa.

[0438] C19. The immunogenic conjugate of any one of C1-C18, wherein the capsular polysaccharide has a molecular weight of between about 25 kDa and about 400 kDa.

[0439] C20. The immunogenic conjugate of any one of C1-C19, wherein the capsular polysaccharide has a molecular weight of between about 25 kDa and about 200 kDa.

[0440] C21. The immunogenic conjugate of any one of C1-C20, wherein the capsular polysaccharide has a molecular weight of between about 100 kDa and about 400 kDa.

[0441] C22. The immunogenic conjugate of any one of C1-21, wherein the molecular weight of the conjugate is between about 300 kDa and about 20,000 kDa.

[0442] C23. The immunogenic conjugate of any one of C1-C22, wherein the molecular weight of the conjugate is between about 1,000 kDa and about 15,000 kDa.

[0443] C24. The immunogenic conjugate of any one of C1-C23, wherein the molecular weight of the conjugate is between about 1,000 kDa and about 10,000 kDa.

[0444] C25. The immunogenic conjugate of any one of C1-C24, wherein each polysaccharide is independently conjugated to a carrier protein.

[0445] C26. Carrier protein is CRM 197or tetanus toxoid.

[0446] C27. Carrier protein is CRM 197 The immunogenic conjugate of any one of C1 to C26,

[0447] C28. A method for isolating capsular polysaccharide, comprising reacting an organic reagent with a cell broth containing a capsular polysaccharide-producing bacterium.

[0448] C29. A method of C28 that does not lyse bacteria.

[0449] C30. Killing bacteria by heat, method C28 or C29.

[0450] C31. The method of any one of C28-C30, further comprising the step of centrifuging to provide a cell paste.

[0451] C32. The method of any one of C28 to C31, further comprising a filtering step.

[0452] C33. The method of C32, wherein said filtering step is hemodiafiltration.

[0453] C34. The method of C28, wherein the bacterium is Streptococcus agalactiae.

[0454] C35. An immunogenic composition comprising the immunogenic polysaccharide-protein conjugate of any one of C1 to C27.

[0455] C36. An immunogenic composition comprising a polysaccharide-protein conjugate, said conjugate comprising capsular polysaccharide from Group B Streptococcus (GBS) serotype VI conjugated to a carrier protein.

[0456] C37. An immunogenic composition comprising one or more polysaccharide-protein conjugates, said conjugates comprising capsular polysaccharides from Group B Streptococcus (GBS) serotype VI and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, IV, V, VII, VIII and IX.

[0457] C38. The immunogenic composition of C37, wherein at least one additional serotype is Ia.

[0458] C39. The immunogenic composition of C38, further comprising a conjugate comprising capsular polysaccharide from GBS serotype Ib.

[0459] C40. An immunogenic composition of C38 or C39, further comprising a conjugate comprising capsular polysaccharide from GBS serotype II.

[0460] C41. The immunogenic composition of any one of C38-C40, further comprising a conjugate comprising capsular polysaccharide from GBS serotype III.

[0461] C42. The immunogenic composition of any one of C38-C41, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IV.

[0462] C43. The immunogenic composition of any one of C38-C42, further comprising a conjugate comprising capsular polysaccharide from GBS serotype V.

[0463] C44. The immunogenic composition of any one of C38-C43, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0464] C45. The immunogenic composition of any one of C38-C44, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0465] C46. The immunogenic composition of any one of C38 to C45, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0466] C47. The immunogenic composition of C36, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VII.

[0467] C48. An immunogenic composition of C36 or C47, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0468] C49. The immunogenic composition of any one of C46-C48, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0469] C50. The immunogenic composition of C37, wherein at least one additional serotype is VII.

[0470] C51. An immunogenic composition of C50, further comprising a conjugate comprising capsular polysaccharide from GBS serotype VIII.

[0471] C52. The immunogenic composition of any one of C50 or C51, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0472] C53. The immunogenic composition of C38, wherein the at least one additional serotype is VIII.

[0473] C54. The immunogenic composition of C53, further comprising a conjugate comprising capsular polysaccharide from GBS serotype IX.

[0474] C55. The immunogenic composition of C54, wherein at least one additional serotype is IX.

[0475] C56. An immunogenic composition comprising a polysaccharide-protein conjugate comprising at least four GBS capsular polysaccharide serotypes selected from the group consisting of Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

[0476] C57. The immunogenic composition of C56, comprising at least two GBS capsular polysaccharide serotypes.

[0477] C58. The immunogenic composition of C56, comprising at least three GBS capsular polysaccharide serotypes.

[0478] C59. The immunogenic composition of C56, comprising at least four GBS capsular polysaccharide serotypes.

[0479] C60. An immunogenic composition of C56, comprising at least five GBS capsular polysaccharide serotypes.

[0480] C61. An immunogenic composition of C56, comprising at least six GBS capsular polysaccharide serotypes.

[0481] C62. An immunogenic composition of C56, comprising at least seven GBS capsular polysaccharide serotypes.

[0482] C63. The immunogenic composition of C56, comprising at least eight GBS capsular polysaccharide serotypes.

[0483] C64. An immunogenic composition of C56, comprising at least nine GBS capsular polysaccharide serotypes.

[0484] C65. The immunogenic composition of any one of C35-C64, further comprising a pharma- ceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic surfactant, inhibitor of free radical oxidation, carrier, or a mixture thereof.

[0485] C66. The immunogenic composition of any one of C35-C65, further comprising a buffering agent.

[0486] C67. The immunogenic composition of C66, wherein the buffer is selected from the group consisting of HEPES, PIPES, MES, Tris(trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate.

[0487] C68. The immunogenic composition of C67, wherein the buffering agent is histidine.

[0488] C69. The immunogenic composition of any one of C35-C68, further comprising a surfactant.

[0489] C70. The immunogenic composition of C69, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20 and polyoxyethylene alkyl ethers.

[0490] C71. The immunogenic composition of C70, wherein the surfactant is polysorbate-80.

[0491] C72. The immunogenic composition of any one of C35-C71, further comprising an excipient.

[0492] C73. The immunogenic composition of C72, wherein the excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water and ethanol.

[0493] C74. The immunogenic composition of C73, wherein the excipient is sodium chloride.

[0494] C75. The immunogenic composition of any one of C35 to C74, further comprising an adjuvant.

[0495] C76. The immunogenic composition of C75, wherein the adjuvant is an aluminum-based adjuvant or QS-21.

[0496] C77. The immunogenic composition of C76, wherein the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyl phosphate and aluminum hydroxide.

[0497] C78. The immunogenic composition of C77, wherein the adjuvant is aluminum phosphate.

[0498] C79. The immunogenic composition of C78, ​​wherein the adjuvant is aluminum hydroxyl phosphate.

[0499] C80. The immunogenic composition of any one of C35-C79, comprising a buffer, a surfactant, an excipient, and optionally an adjuvant, and buffered to a pH of about 6.0 to about 7.0.

[0500] C81. The immunogenic composition of any one of C35-C80, comprising histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, buffered to a pH of about 6.0 to about 7.0.

[0501] C82. The immunogenic composition of any one of C35-C81, comprising about 10 mM to about 25 mM histidine, about 0.01% to about 0.03% (v / w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml aluminum as aluminum phosphate.

[0502] C83. The immunogenic composition of any one of C35-C82, comprising a dose of about 5 mcg / ml to about 50 mcg / ml.

[0503] C84. The immunogenic composition of any one of C35-C83, optionally in the presence of at least one excipient, which is lyophilized.

[0504] C85. The immunogenic composition of C84, wherein at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, wheat flour, stone flour, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerol, propylene glycol, water, and ethanol.

[0505] C86. The immunogenic composition of C85, wherein at least one excipient is sucrose.

[0506] C87. The immunogenic composition of any one of C84-C86, comprising about 1% (w / v) to about 10% (w / v) of at least one excipient.

[0507] C88. The immunogenic composition of any one of C84-C87, comprising an additional excipient.

[0508] C89. The immunogenic composition of C88, wherein the additional excipient is mannitol or glycine.

[0509] C90. The immunogenic composition of C88 or C89, comprising about 1% (w / v) to about 10% (w / v) of an additional excipient.

[0510] C91. The immunogenic composition of any one of C84-C90, which is reconstituted with water, water for injection (WFI), an adjuvant suspension or saline.

[0511] C92. The immunogenic composition of any one of C44 to C91 for use as a medicament.

[0512] C93. The immunogenic composition of any one of C35 to C92 for use in a method of inducing an immune response to GBS in a subject.

[0513] C94. The immunogenic composition of C93, wherein the subject is a woman planning pregnancy or who is pregnant.

[0514] C95. The immunogenic composition of C94, wherein the female is in the second half of pregnancy.

[0515] C96. The immunogenic composition of C95, wherein the pregnant female is at least 20 weeks pregnant.

[0516] C97. The immunogenic composition of C96, wherein the pregnant female is between 27 and 36 weeks pregnant.

[0517] C98. The immunogenic composition of C97, wherein the subject is an adult 50 years of age or older.

[0518] C99. The immunogenic composition of C98, wherein the subject is an adult aged 65 years or older.

[0519] C100. The immunogenic composition of C99, wherein the subject is an adult aged 85 years or older.

[0520] C101. The immunogenic composition of any one of C90 to C100, wherein the subject is immunocompromised.

[0521] C102. The immunogenic composition of C101, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

[0522] C103. The immunogenic composition of any one of C93 to C102, wherein the group B streptococcus is Streptococcus agalactiae.

[0523] C104. A method of inducing an immune response against group B streptococcus, comprising administering to a subject an effective amount of the immunogenic composition of any one of C35 to C103.

[0524] C105. A method of preventing or reducing a disease or condition associated with Group B Streptococcus in a subject, comprising administering to the subject an effective amount of an immunogenic composition of any one of C35 to C104.

[0525] C106. The method of C104 or C105, wherein the subject is a woman planning a pregnancy or a pregnant woman.

[0526] C107. The method of C106, wherein the woman is in the second half of pregnancy.

[0527] C108. The method of C106 or C107, wherein the pregnant female is at least 20 weeks pregnant.

[0528] C109. The method of any one of C106 to C108, wherein the pregnant woman is between 27 and 36 weeks pregnant.

[0529] C110. The method of C104 or C105, wherein the subject is an adult aged 50 years or older.

[0530] C111. The method of C110, wherein the subject is an adult aged 65 years or older.

[0531] C112. The method of C110 or C111, wherein the subject is an adult aged 85 years or older.

[0532] C113. The method of any one of C104 to C112, wherein the subject is immunocompromised.

[0533] C114. The method of C113, wherein the subject has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease or liver disease.

[0534] C115. The method of any one of the preceding paragraphs, wherein the group B streptococcus is Streptococcus agalactiae.

[0535] C116. An antibody that binds to the capsular polysaccharide in the immunogenic conjugate of any one of C1 to C27.

[0536] C117. A composition comprising the antibody of C116.

[0537] C118. A method for conferring passive immunity to a subject, comprising: (a) producing an antibody preparation using the immunogenic composition of any preceding paragraph; (b) administering the antibody preparation to a subject to confer passive immunity; The method includes:

[0538] C119. A method for producing an immunogenic polysaccharide-protein conjugate according to any one of C1 to C27, comprising: (a) reacting GBS capsular polysaccharide with an oxidizing agent to provide an activated polysaccharide; (b) reacting the activated polysaccharide with a carrier protein to provide a polysaccharide-protein conjugate; The method includes:

[0539] C120. The method of C119, wherein step (b) is carried out in a polar aprotic solvent.

[0540] C121. The method of C120, wherein the solvent is selected from the group consisting of dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF) and hexamethylphosphoramide (HMPA).

[0541] C122. The method of C121, wherein the solvent is dimethylsulfoxide (DMSO).

[0542] C123. The method of any one of C119-C122, wherein the polysaccharide is reacted with 0.01 to 10.0 molar equivalents of an oxidizing agent.

[0543] C124. The method of any one of C119-C123, wherein the oxidizing agent is a periodate.

[0544] C125. The method of C124, wherein the periodate is sodium periodate.

[0545] C126. The process of any one of C119-C125, wherein the oxidation reaction of step (a) is for between 1 hour and 50 hours.

[0546] C127. The method of any one of C119-C126, wherein the temperature of the oxidation reaction is maintained between about 2° C. and about 25° C.

[0547] C128. The method of any one of C119-C127, 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.

[0548] C129. The method of C128, wherein the buffer has a concentration of between about 1 mM and about 500 mM.

[0549] C130. The method of any one of C119-C129, wherein the oxidation reaction is carried out at a pH between about 4.0 and about 8.0.

[0550] C131. The method of C119, wherein the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO).

[0551] C132. The method of C131, wherein N-chlorosuccinimide (NCS) is the co-oxidant.

[0552] C133. The method of any one of C119 to C132, wherein step (a) further comprises quenching the oxidation reaction by addition of a quenching agent.

[0553] C134. The method of any one of C119-C133, wherein the concentration of the polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL.

[0554] C135. The method of any one of C119 to C134, wherein the degree of oxidation of the activated polysaccharide is between 5 and 25.

[0555] C136. The method of any one of C119-C135, further comprising the step of lyophilizing the activated polysaccharide.

[0556] C137. The method of C136, wherein the activated polysaccharide is lyophilized in the presence of a sugar selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit.

[0557] C138. Step (b) is (1) combining an activated polysaccharide with a carrier protein; (2) reacting the combined activated polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; The method of any one of C119 to C137, comprising:

[0558] C139. The method of C138, wherein the concentration of the activated polysaccharide in step (2) is between about 0.1 mg / mL and about 10.0 mg / mL.

[0559] C140. The method of C137 or C138, wherein the initial ratio of activated polysaccharide to carrier protein (wt / wt) is between 5:1 and 0.1:1.

[0560] C141. The reducing agent is a Bronsted or Lewis acid, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i The process of any one of C138 to C139, wherein the cation is selected from the group consisting of sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride or zinc in the presence of PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB).

[0561] C142. The method of C141, wherein the reducing agent is sodium cyanoborohydride.

[0562] C143. The method of any one of C138-C142, wherein the amount of reducing agent is between about 0.1 and about 10.0 molar equivalents.

[0563] C144. The method of any one of C138 to C143, wherein the duration of the reduction reaction in step (2) is between 1 hour and 60 hours.

[0564] C145. The method of any one of C138-C144, wherein the temperature of the reduction reaction is maintained between 10° C. and 40° C.

[0565] C146. The method of any one of C119-C145, further comprising the step of capping any unreacted aldehyde by the addition of a borohydride (step (c)).

[0566] C147. The process of C146, wherein the amount of borohydride is between about 0.1 and about 10.0 molar equivalents.

[0567] C148. The process of C146, wherein the borohydride is selected from the group consisting of sodium borohydride (NaBH4), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride and magnesium borohydride.

[0568] C149. The method of C146, wherein the borohydride is sodium borohydride (NaBH4).

[0569] C150. The method of C146, wherein the duration of the capping step is between 0.1 hours and 10 hours.

[0570] C151. The method of any one of C146-C149, wherein the temperature of the capping step is maintained between about 15° C. and about 45° C.

[0571] C152. The method of any one of C119-C151, further comprising purifying the polysaccharide-protein conjugate.

[0572] C153. The method of any one of C119-C152, wherein the polysaccharide-protein conjugate comprises less than about 40% free polysaccharide compared to the total amount of polysaccharide.

[0573] C154. The method of any one of C119-C153, wherein the ratio of polysaccharide to carrier protein (weight / weight) in the conjugate is between about 0.5 and about 3.0.

[0574] C155. The method of any one of C119-C154, wherein the degree of conjugation of the conjugate is between 2 and 15.

[0575] C156. A method for producing a polysaccharide-protein conjugate, comprising: (a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; (b) quenching the oxidation reaction of step (a) by the addition of a quenching agent to provide activated GBS capsular polysaccharide; (c) combining the activated GBS capsular polysaccharide with a carrier protein; (d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate; (e) capping any unreacted aldehyde by addition of sodium borohydride (NaBH4); Including, wherein steps (c) and (d) are carried out in DMSO.

[0576] References 1. Paoletti, LJ, J. Bradford, and LC Paoletti, "A Serotype VIII Strain among Colonizing Group B Streptococcal Isolates in Boston, Massachusetts." "Isolates in Boston, Massachusetts.)" Journal of Clinical Microbiology, 1999. 37(11): p. 3759-3760. 2. Lachenauer, CS et al., "Serotypes VI and VIII predominate among group B streptococci isolated from pregnant Japanese women." isolated from pregnant Japanese women. )”J Infect Dis, 1999. 179(4): p. 1030-3. 3. Matsubara, K. et al., Group B streptococcal disease in infants in the first year of life: A nationwide surveillance study in Japan, 2011-2015. a nationwide surveillance study in Japan, 2011-2015.)” Infection, 2017. 25: p. 25. 4. Chang, B. et al., Characteristics of group B Streptococcus isolated from infants with invasive infections: a population-based study in Japan. invasive infections: a population-based study in Japan.)” Japanese Journal of Infectious Diseases, 2014. 67(5): p. 356-60. 5. Lu, B. et al., "Molecular characteristics and antimicrobial resistance in invasive and noninvasive group B streptococci in China between 2008 and 2015." and noninvasive Group B Streptococcus between 2008 and 2015 in China.)” Diagnostic Microbiology & Infectious Disease, 2016. 86(4): p. 351-357. 6. Campisi, E. et al., Genomic Analysis Reveals Multi-Drug Resistance Clusters in Group B Streptococcus CC17 Hypervirulent Isolates Causing Neonatal Invasive Disease in Southern Mainland China. Streptococcus CC17 Hypervirulent Isolates Causing Neonatal Invasive Disease in Frontiers in Microbiology, 2016. 7: p. 1265. 7. Liu, H. et al., "Estimating the burden of invasive Group B Streptococcal disease in young infants in southern mainland China: an observational study." young infants in southern mainland China: An observational study.)” International Journal of Clinical and Experimental Medicine, 2015. 8(8): p.13699-13707. 8. Morozumi, M. et al., "Molecular characteristics of Group B streptococci isolated from adults with invasive infections in Japan." adults with invasive infections in Japan.)” Journal of Clinical Microbiology, 2016. 24: p. 24. 9. Tsai, M.-H. et al., "Molecular Characteristics and Antimicrobial Resistance of Group B Streptococcus Strains Causing Invasive Disease in Neonates and Adults." Streptococcus Strains Causing Invasive Disease in Neonates and Adults.)” Frontiers in microbiology, 2019. 10: p. 264-264.

Claims

1. 1. An immunogenic composition comprising a polysaccharide-protein conjugate comprising group B streptococcus (GBS) capsular polysaccharide of serotype VI and streptococcal C5a peptidase (SCP) carrier protein for preventing or reducing a disease or condition caused by at least one of group B streptococcus (GBS) serotypes Ia, Ib, II, III, IV, V, VIII or IX.

2. 2. The immunogenic composition of claim 1, wherein the disease or condition is caused by at least one of GBS serotypes III, VIII or IX.

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