Group B Streptococcus polysaccharide-protein conjugates, methods for producing conjugates, immunogenic compositions containing 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 an immunogenic polysaccharide-protein conjugate comprising a capsular polysaccharide (CP) derived 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 greater than approximately 60%. The present invention also relates to a method for preparing the conjugate and an immunogenic composition comprising the conjugate. The present invention also relates to an immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises CP derived from at least one GBS serotype selected from serotypes VI, VII, VIII, and IX, and optionally one or more additional GBS serotypes. The present invention further relates to a method for inducing an immune response to GBS in a subject, and / or for reducing or preventing invasive GBS disease in a subject using the compositions disclosed herein. The antibodies obtained can be used to treat or prevent GBS infection through passive immunotherapy, or to immunize mothers through antibody transfer in the mother for the protection of their offspring. [Background technology]

[0002] Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive, polysaccharide-encapsulated organism. These are common commensal organisms in the human gastrointestinal and reproductive tracts and are a cause of serious illness in infants and the elderly (Baker, CJ, Vaccine, 31(Appendix 4):D3~D6(2013)). The main 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 the newborn before or during childbirth, potentially causing sepsis, pneumonia, and meningitis (Baker 2013; Heath, PT et al., BMJ Clin. Evid. (Online), pii:0323 (2014)). At least 25 percent of infants who overcome GBS meningitis experience neurological impairment, and it is estimated that 19% experience cognitive delay, cerebral palsy, blindness, and hearing loss (Libster, R. et al., Pediatrics, 130(1):e8-152012 (2012)). GBS is also 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 have a much higher risk of infection, with up to 3% becoming infected and up to 30% having a mortality rate even with immediate antibiotic treatment (Heath 2014).

[0003] In the late 1990s, the introduction of GBS screening and intrapartum prophylactic antibiotic administration (IAP) in the United States reduced the rate of neonatal disease (EOD) occurring within the first week of life, but did not have a measurable effect on the rate of late-onset disease (LOD) occurring within the first three months of life. The current rates of EOD and LOD in the United States are 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). Despite the introduction of a pneumococcal conjugate vaccine for the prevention of invasive pneumococcal disease, including bacteremia and meningitis, and despite the introduction of an intra-abstract antiprescription (IAP) for the prevention of GBS disease, GBS remained the single most common cause of neonatal sepsis (EOD) and meningitis in infants (under 2 months of age) in the United States (Verani, JR et al., MMWR, 59(RR10):1~32 (2010); Thigpen, MC et al., New England Journal of Medicine, 364(21):2016~2025 (2011)). Unlike in the United States, the introduction of preventive guidelines for invasive GBS disease and IAP did not reduce the incidence of EOD in either the Netherlands or the United Kingdom (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)). The lack of this effect may be due to the lack of universal screening and the restriction of IAP to mothers at the highest risk (e.g., fever, prolonged membrane rupture). The rate of EOD was significantly higher in countries that did not use IAP, with a mean incidence of 0.75 per 1,000 births (95% CI 0.58–0.89) reported (Edmond, KM et al., Lancet, 379(9815):547–556(2012)).

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

[0005] One approach to preventing GBS disease in infants and the elderly is the use of capsular polysaccharide-based vaccines. Maternal GBS prophylactic vaccination, whether or not IAP is used, has the potential to prevent GBS disease in infants in the United States. While polysaccharides themselves can be immunogenic, conjugation of polysaccharides with protein carriers has been used to improve immunogenicity, particularly in infants and the elderly. Polysaccharide-protein conjugate vaccines are made using polysaccharides, generally derived from bacterial exocophores, associated with the protein carrier. The chemical bond between the polysaccharide and the protein carrier triggers an immune response against bacteria exhibiting the polysaccharide contained within the vaccine on their surface, thus preventing disease. Therefore, vaccination using polysaccharides derived from pathogenic bacteria is a potential strategy to enhance host immunity.

[0006] The structure of polysaccharides covering bacteria varies considerably, even within a single bacterial species. For example, GBS has 10 different serotypes (i.e., serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX) due to variations in the bacterial polysaccharide capsule. Therefore, polysaccharide-based vaccines should ideally consist of a set of polysaccharides that can reliably cover a broad range of different circulating strains.

[0007] The carrier protein may be an associated protein antigen derived from the target pathogen that enhances a specific immune response to 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 for GBS serotypes Ia, Ib, II, III, and V have been evaluated in Phase 1 and 2 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)). This includes 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 studied (Baker JID 2003; Clicaltrials.gov NCT01193920, NCT01412801 and NCT01446289). However, a GBS vaccine has not yet been approved.

[0009] Furthermore, while the trivalent vaccine covers over 90% of the invasive strains causing neonatal disease in South Africa (Madzivhandila, M. et al., PloS One, 6(3):e17861(2011)), these same serotypes represent only 62% and 66% of invasive isolates in North America and Europe, respectively, based on surveillance of recent neonatal isolates from a global collection of 901 samples collected between 2004 and 2013 from the Tigecycline Evaluation and Surveillance Study (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 have 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); Teatro (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); Palmieero, JK et al., Journal of Clinical Microbiology, 48(12):4397~4403 (2010)). A study investigating rectal / vaginal carriage in adults, a risk factor for GBS transmission to infants, found that 97% of isolates belonged to one of these six serotypes, with serotype IV accounting for approximately 4%. The study was designed to monitor carriage of beta-hemolytic streptococci (including 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 elderly individuals aged 65 and older belonged to the same six dominant serogroups. The most significant difference between elderly isolates and other populations was serogroup distribution. For isolates from elderly patients, serotype V constituted the largest group (34% for neonates versus 18% for adult carrier strains).

[0012] Studies in GBS epidemiology have found geographical variance in serotype prevalence, indicating that the prevalence of non-GBS6 serotypes is determined by a number of factors, including topography, patient age, and whether surveillance is based on established or invasive disease.

[0013] For example, serotypes VI and VIII isolates have been shown to be predominantly exotic 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 correspondingly cause high disease rates in infants. Large-scale GBS epidemiological studies in Japan collected between 2011 and 2015 (n= In 132), the prevalence of non-GBS6 serotypes was very low (1.1% for serotype VI in EOD only; 1.1% for serotype IX in late-onset disease only)[3]. These results are consistent with the early neonatal invasive isolate study (n=60), which showed similar serotype prevalences: III (48.3%), Ia (30.0%), and Ib (10.0%)[4]. This trend for low rates of non-GBS6 neonatal disease appears to be similar to that in China, although studies published to date have involved fewer 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 the elderly but not in infants [9].

[0015] Based on these surveillance studies, serotype VI appears to be an emerging threat that promotes inclusion in second-generation vaccines, particularly those targeting the elderly. The incidence of invasive diseases caused by serotypes VII, VIII, and IX is currently rare, but could become more important if serotype replacement occurs after the introduction of the GBS6 vaccine.

Summary of the Invention

Problems to be Solved by the Invention

[0016] Therefore, there is a need for a polysaccharide-protein conjugate vaccine or a monoclonal antibody to confer passive immunity as a means to prevent or treat GBS diseases, including those caused by the newly emerging serotypes VI, VII, VIII, and IX, among a wide range of populations worldwide.

Means for Solving the Problems

[0017] In one embodiment, the invention includes an immunogenic composition comprising a polysaccharide-protein conjugate comprising a group B streptococcus (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%. In another embodiment, the invention includes 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 of sialic acid per mM of polysaccharide.

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

[0020] In another embodiment, the present invention includes an immunogenic composition as described herein, wherein the conjugate has a molecular weight 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 a further embodiment, the present invention includes an immunogenic composition as described herein, wherein the capsular polysaccharide is O-acetylated at less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%.

[0022] In another embodiment, the present invention includes an immunogenic composition as described herein, 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 of O-acetate per mM of sugar repeating unit.

[0023] In one embodiment, the present invention, the carrier protein is CRM 197 , diphtheria toxoid (DT), tetanus toxoid (TT), and streptococcal C5a peptidase (SCP), and includes an immunogenic composition as described herein.

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

[0025] In another embodiment, the present invention includes a method for isolating a capsular polysaccharide as described, which does not lyse bacteria and / or kills bacteria by heat.

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

[0027] In another embodiment, the present invention includes a method for isolating a capsular polysaccharide as described, further comprising a filtering step which may include a filtering step which is hemodiafiltration.

[0028] In yet another embodiment, the present invention includes a method for isolating the described capsular polysaccharides, including Streptococcus agalactiae, using a capsular polysaccharide-producing fungus.

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

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

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

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

[0033] In one embodiment, the present invention comprises an immunogenic composition comprising a capsular polysaccharide-protein conjugate prepared by the method described herein.

[0034] In another embodiment, the present invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate as described herein, wherein the conjugate comprises a capsular polysaccharide derived from serotype VI and carrier protein of Group B Streptococcus (GBS), 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 present invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VI and a carrier protein. In another embodiment, the present 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 present invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate containing 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 present invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate containing GBS serotype VI capsular polysaccharide, further comprising at least one additional serotype selected from VII, VIII, and IX.

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

[0038] In another embodiment, the present invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VII and a carrier protein. In another embodiment, the present 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 another embodiment, the present invention comprises 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, and the capsular polysaccharide has a sialic acid level of more than 60%, more than 95%, or more than 100%.

[0040] In another embodiment, the present invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype VIII and a carrier protein. In another embodiment, the present 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 present invention comprises 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, and the capsular polysaccharide has a sialic acid level of more than 60%, more than 95%, or more than 100%.

[0042] In another embodiment, the present invention comprises an immunogenic composition comprising a polysaccharide-protein conjugate comprising GBS capsular polysaccharide serotype IX and a carrier protein. In another embodiment, the present 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 further embodiments, the present invention comprises 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, and the capsular polysaccharide has a sialic acid level of more than 60%, more than 95%, or more than 100%.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0064] In another embodiment, the present invention includes immunogenic compositions as described herein, further comprising pharmaceutically acceptable excipients, buffers, stabilizers, adjuvants, antifreeze agents, salts, divalent cations, nonionic surfactants, free radical oxidation inhibitors, carriers, or mixtures thereof.

[0065] In yet another embodiment, the present 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 present invention includes 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 one embodiment, the present 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, melegitose, dextran, mannitol, lactitol, palatinite, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim milk powder, glycerol, propylene glycol, water, and ethanol.

[0068] In another embodiment, the present invention further comprises an immunogenic composition as described herein, comprising Streptococcus C5a peptidase (SCP), an aluminum-based adjuvant, or an adjuvant selected from 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 present 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 another embodiment, the present 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 present invention includes 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 further embodiments, the present invention includes immunogenic compositions as described herein, comprising doses ranging from about 5 mcg / ml to about 50 mcg / ml.

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

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

[0075] In another embodiment, the present 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 one embodiment, the present invention includes an immunogenic composition as described herein, which is redissolved in water, water for injection (WFI), an adjuvant suspension, or physiological saline.

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

[0078] In yet another embodiment, the present invention includes an immunogenic composition as described herein for use in a method for inducing an immune response to GBS in a subject.

[0079] In another embodiment, the present invention relates to a woman who is planning to become pregnant or is pregnant, and the woman is in the second half of her pregnancy, at least 20 weeks pregnant, or 27 to 36 weeks pregnant, and includes an immunogenic composition as described herein.

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

[0081] In one embodiment, the present invention includes an immunogenic composition as described herein, wherein the subject is immunodeficient and / or 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 present invention comprises an immunogenic composition as described herein, wherein the Group B Streptococcus is Streptococcus agalactiae.

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

[0084] In another embodiment, the present invention includes a method for 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 present invention provides a method for 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 to become pregnant or a pregnant woman, and optionally the woman is in the second half of pregnancy, at least 20 weeks pregnant, or 27 to 36 weeks pregnant.

[0086] In another embodiment, the present invention provides a method for 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 one embodiment, the present invention provides a method for 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 present invention includes a method for inducing an immune response to serotype V, serotype VI, serotype VII, serotype VIII, or serotype IX of Group B Streptococcus, comprising the step of administering to a subject an immunogenic composition as described herein.

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

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

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

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

[0093] In one embodiment, the present invention provides a method for preparing an immunogenic polysaccharide-protein conjugate as described herein, comprising reacting a polysaccharide with 0.01 to 10.0 molar equivalents of an oxidizing agent, wherein the oxidizing agent is a periodate, which may contain sodium periodate.

[0094] In another embodiment, the present invention includes a method for producing an immunogenic polysaccharide-protein conjugate as described herein, wherein the oxidation reaction in step (a) is carried out for a period of 1 to 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 the buffer may have a concentration between about 1 mM and about 500 mM.

[0095] In another embodiment, the present invention relates to a method for producing 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 a step of further quenching the oxidizing reaction product by adding a quenching agent.

[0096] In another embodiment, the present invention includes a method for preparing 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 is between 5 and 25.

[0097] In yet another embodiment, the present invention includes a method for producing an immunogenic polysaccharide-protein conjugate as described herein, further comprising the step of freeze-drying an activated polysaccharide in the presence of a sugar selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melegitose, dextran, mannitol, lactitol, and palatinite.

[0098] In further embodiments, the present invention includes a method for preparing an immunogenic polysaccharide-protein conjugate as described herein, wherein step (b) comprises the steps of: compounding an activated polysaccharide with a carrier protein; and reacting the compounded activated polysaccharide and carrier protein with a reducing agent to form a GBS capsule 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 (weight / weight) of the activated polysaccharide to the carrier protein may be between 5:1 and 0.1:1.

[0099] In further embodiments, the present invention relates to a method for preparing immunogenic polysaccharide-protein conjugates as described herein, wherein the reducing agent is a Brønsted or Lewis acid, pyridineborane, 2-picolinborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i The method includes a reducing agent selected from the group consisting of sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride, and zinc, and / or the amount of the reducing agent is between about 0.1 and about 10.0 molar equivalents, in the presence of PrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridineborane (PEMB).

[0100] In one embodiment, the present invention includes a method for producing 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 further embodiments, the present invention includes a method for preparing an immunogenic polysaccharide-protein conjugate as described herein, further comprising a step of capping unreacted aldehydes by adding boron hydride (step (c)), wherein the amount of boron hydride may be between about 0.1 and about 10.0 molar equivalents, and the boron hydride is selected from the group consisting of sodium borohydride (NaBH4), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, and magnesium borohydride, and the duration of the capping step may be between 0.1 and 10 hours, and / or the temperature of the capping step is maintained between about 15°C and about 45°C.

[0102] In another embodiment, the present invention includes a method for preparing an immunogenic polysaccharide-protein conjugate as described herein, wherein the polysaccharide-protein conjugate contains less than 40% free polysaccharides relative to the total amount of polysaccharides.

[0103] In another embodiment, the present invention includes a method for preparing a polysaccharide-protein conjugate as described herein, wherein the ratio (weight / weight) of polysaccharide to carrier protein 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 present invention provides a method for preparing a polysaccharide-protein conjugate as described herein, comprising: (a) reacting isolated GBS capsule polysaccharide with an oxidizing agent; (b) quenching the oxidizing reaction product from step (a) by adding a quenching agent to obtain an activated GBS capsule polysaccharide; (c) compounding the activated GBS capsule polysaccharide with a carrier protein; (d) reacting the compounded activated GBS capsule polysaccharide and carrier protein with a reducing agent to form a GBS capsule polysaccharide-carrier protein conjugate; and (e) capping unreacted aldehydes by adding sodium borohydride (NaBH4), wherein steps (c) and (d) are carried out in DMSO.

[0105] In one embodiment, the present invention provides a method for preparing a polysaccharide-protein conjugate as described herein, comprising: (a) reacting isolated GBS capsule polysaccharide with an oxidizing agent; (b) quenching the oxidizing reaction product from step (a) by adding a quenching agent to obtain an activated GBS capsule polysaccharide; (c) compounding the activated GBS capsule polysaccharide with a carrier protein; (d) reacting the compounded activated GBS capsule polysaccharide and carrier protein with a reducing agent to form a GBS capsule polysaccharide-carrier protein conjugate; (e) capping unreacted aldehydes by adding sodium borohydride (NaBH4); and (f) purifying the polysaccharide-protein conjugate, wherein steps (c) and (d) are carried out in DMSO. [Brief explanation of the drawing]

[0106] [Figure 1] This figure shows the immunogenicity and cross-reactivity of GBS capsular polysaccharide serotype VI conjugate. [Figure 2A] This figure shows the immunogenicity of GBS capsular polysaccharide serotype VII conjugate. [Figure 2B] This figure shows the immunogenicity of GBS capsular polysaccharide serotype VIII conjugate. [Figure 2C] This figure shows the immunogenicity of GBS capsular polysaccharide serotype IX conjugate. [Figure 3A] This figure shows the immunogenicity and cross-reactivity of GBS capsular polysaccharide serotype VII conjugate. [Figure 3B] This figure shows the immunogenicity and cross-reactivity of GBS capsular polysaccharide serotype IX conjugate. [Figure 4] This figure shows the immunogenicity of the polyvalent GBS conjugate vaccine. [Modes for carrying out the invention]

[0107] This invention is not limited to the specific methods and experimental conditions described, and such methods and conditions may be modified. It should also be understood that the technical terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

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

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

[0110] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” encompass multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to “the method” encompasses one or more methods and / or steps described herein and / or what would be expected to be apparent to a person skilled in the art by reading this disclosure, etc.

[0111] The terms “approximately” or “about” mean a statistically meaningful range of values. Such a range may be within one decimal place, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The permissible deviation covered by the terms “approximately” or “about” is determined by the particular system under study and will be readily apparent to those skilled in the art. Whenever a range is described in this application, all integers within that range are also contemplated as embodiments of the invention.

[0112] In this disclosure, terms such as “include,” “contain,” “contain,” “include,” “contain,” and “contain,” may have meanings belonging to U.S. patent law, for example, they may mean “inclusive,” “included,” and “inclusive.” Such terms refer to the inclusion of a particular ingredient or set of ingredients without excluding any other elements. Terms such as “essentially from” and “essentially from” have meanings belonging to U.S. patent law, for example, allowing for the inclusion of additional ingredients or steps that do not deviate from the novel or fundamental features of the invention, i.e., excluding additional unlisted ingredients or steps that deviate from the novel or fundamental features of the invention, and excluding prior art ingredients or steps, such as when the aim of this document is to define patentable embodiments, e.g., novel, non-obvious, inventive, and superior to the prior art, e.g., documents cited or incorporated herein by reference. Also, terms such as “consisting of” and “consisting of” have meanings belonging to U.S. patent law, i.e., these terms are closed-ended. Therefore, these terms refer to the inclusion of a specific raw material or set of raw materials and the exclusion of all other raw materials.

[0113] The term “antigen” generally refers to an immunogenic substance that can stimulate the production of an antibody or T-cell response, or both, in an animal, which is typically a protein, peptide, polysaccharide, lipid, or conjugate, or in some cases, a composition injected or absorbed into an animal, containing at least one epitope to which a congener antibody can selectively bind. The immune response may be produced against the entire molecule or against one or more different parts of the molecule (e.g., an epitope or hapten). The term may be used to refer to a homogeneous or heterogeneous population of individual molecules or antigen molecules. Antigens are recognized by antibodies, T-cell receptors, or other elements of specific humoral and / or cellular immunity. The term “antigen” encompasses all relevant antigenic epitopes. The epitopes of a given antigen can be identified using several epitope mapping techniques well known to those skilled in the art (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (edited by Glenn E. Morris, 1996), Humana Press, Totowa, NJ). For example, linear epitopes can be determined by simultaneously synthesizing a number of peptides on a solid support, peptides corresponding to portions of protein molecules, and reacting the peptides with an antibody while the peptides remain attached to the support. Such techniques are publicly known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen, H.M. et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1984); Geysen, H.M. et al., Molec. Immunol., 23(7):709-715 (1986), all of which are incorporated herein by reference in their entirety. Similarly, structural epitopes can be identified by determining the spatial structure of amino acids, for example by X-ray crystallography and two-dimensional nuclear magnetic resonance (e.g., Epitope Mapping Protocols, see above).Furthermore, for the purposes of this invention, “antigen” may also be used to refer to proteins (generally conserved, but may be non-conserved) that include modifications to their native sequence, such as deletions, additions, and substitutions, as long as the protein maintains its ability to produce an immunological response. These modifications may be intentional, such as through site-directed mutagenesis, specific synthetic procedures, or genetic engineering approaches, or they may be accidental, such as through mutations in the host that produces the antigen. Furthermore, antigens may be derived, obtained, or isolated from microorganisms, such as bacteria, or they may be whole organisms. Similarly, oligonucleotides or polynucleotides expressing antigens, such as in nucleic acid immunization applications, are also included in the definition. This also includes synthetic antigens, such as polyepitopes, adjacent epitopes, and other recombinant or synthetically induced antigens (Bergmann, C. et al., Eur. J. Immunol., 23(11):2777~2781 (1993); Bergmann, C. 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” refer to a pharmaceutical composition comprising at least one immunogenic composition that is interchangeable and induces an immune response in an animal.

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

[0116] As used herein, "monosaccharide" refers to a single sugar residue in an oligosaccharide. As used herein, "disaccharide" refers to a polysaccharide consisting of two monosaccharide units or parts 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. In the context of oligosaccharides, individual monomer units or moieties are monosaccharides that are or can be bonded 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 decomposition of biologically produced polysaccharides. Alternatively, oligosaccharides may 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 parts. For clarity, a greater number of repeating units (where n is greater than about 5, e.g., greater than about 10, etc.) are referred to herein as polysaccharides.

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

[0120] In one embodiment, the polysaccharide is a capsular polysaccharide. A capsular polysaccharide is a glycopolymer containing repeating units of one or more monosaccharides linked by glycosidic bonds. Capsular polysaccharides typically form a capsule-like layer around bacterial cells. "Capsular polysaccharide" or "capsule polysaccharide" refers to the polysaccharide capsule outside the cell wall of most Streptococcal isolates. For example, all GBS capsular polysaccharides have branched repeating structures with terminal α2-3-linked sialic acid required for bacterial activity. Capsule-associated sialic acid (quantified by HPLC assay) was detected in over 94% of invasive neonatal isolates from TEST cultured in vitro.

[0121] The inventors have discovered that the sialic acid level of GBS capsular polysaccharides is a crucial feature for generating an immune response. Prior disclosures have provided conflicting information regarding sialic acid levels for serotype V, only the finding that desialylated serotype V is preferred (International Patent Application Publication WO2012 / 035519) and that sialic acid content exceeding 50% may be used for serotype V (International Patent Application Publication WO2014 / 053612). However, none of these references describe the importance of sialic acid levels for at least the majority of GBS polysaccharides to immunogenicity. Surprisingly, the inventors have found that GBS capsular polysaccharides require at least 60% sialic acid before conjugation to provide an immune response equivalent to that of polysaccharides with naturally occurring sialic acid levels (i.e., 100% or about 95%). Even at a sialic acid level of 58%, which is within the previously disclosed range for serotype V, it adversely affected immunogenicity.

[0122] Therefore, in one embodiment of the present invention, the capsular polysaccharides contain their natural sialic acid levels, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharides may be desiallated to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), and up to about 5% (sialylation level of more than about 95%).

[0123] It should be noted that a 100% sialic acid level corresponds to approximately 1.0 mM sialic acid per mM polysaccharide. Therefore, capsular polysaccharides may contain approximately 1.0 mM sialic acid per mM polysaccharide, for example, at least approximately 0.95 mM sialic acid per mM polysaccharide. In further embodiments, the capsule polysaccharide may contain at least about 0.6 mM sialic acid per 1 mM of polysaccharide, for example, at least about 0.65 mM sialic acid per 1 mM of polysaccharide, at least about 0.7 mM sialic acid per 1 mM of polysaccharide, at least about 0.75 mM sialic acid per 1 mM of polysaccharide, at least about 0.8 mM sialic acid per 1 mM of polysaccharide, at least about 0.85 mM sialic acid per 1 mM of polysaccharide, at least about 0.9 mM sialic acid per 1 mM of polysaccharide, or at least about 0.95 mM sialic acid per 1 mM of polysaccharide.

[0124] The terminal sialyl 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%), while serotypes Ia, II, and VII have little to no O-acetylation (less than about 5%) (Lewis 2004). In one embodiment, the capsular polysaccharides include their natural O-acetylation levels (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 Publications WO2005 / 033148 and WO00 / 56357). Another commonly used method is described by Hestrin, S., J. Biol. Chem., 180:249~261 (1949).

[0125] Furthermore, 100% O-acetate corresponds to approximately 1.0 mM O-acetate per mM sugar repeating unit. Therefore, partially O-acetylated polysaccharides contain at least approximately 0.1, 0.2, 0.3, 0.35, or approximately 0.4 mM O-acetate per mM sugar repeating unit. O-deacetylated polysaccharides contain less than approximately 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM sugar repeating unit.

[0126] Streptococcal microorganisms that can cause invasive diseases can generally also produce capsular polysaccharides (CPs) that encase the bacteria and enhance their resistance to clearance by the host's innate immune system. CPs help to cover bacterial cells within a protective capsule that makes bacteria resistant to phagocytosis and intracellular death. Bacteria lacking a capsule are more susceptible to phagocytosis. Capsular polysaccharides are important bacterial dynamics factors for many bacterial pathogens, frequently including Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Staphylococcus aureus.

[0127] Capsular polysaccharides can be used to determine the serotype of specific bacterial species. Typing of the specific structure or unique epitope features of the capsular polysaccharide is usually performed by reaction with a specific antiserum or monoclonal antibody 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 present invention, the polysaccharide is isolated from Streptococcus agalactiae. The polysaccharide is isolated from any encapsulated strain of S. agalactiae, e.g., 090, A909 (ATCC accession number BAA-1138), 515 (ATCC accession number BAA-1177), B523, CJB524, MB4052 (ATCC accession number 31574), H36B (ATCC accession number 12401), S40, S42, MB4053 (ATCC accession number 31575), M709, 133, 7 357, PFEGBST0267, MB4055 (ATCC accession number 31576), 18RS21 (ATCC accession number BAA-1175), S16, S20, V8 (ATCC accession number 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB4082 (ATCC accession number 31577), M132, 110, M781 (ATCC accession number BAA-22), D136C(3) (ATCC C accession number 12403), M782, S23, 120, MB4316 (M-732; ATCC accession number 31475), M132, K79, COH1 (ATCC accession number BAA-1176), PFEGBST0563, 3139 (ATCC accession number 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession number BAA-23), CJB112, 2603V / It can be isolated from R (ATCC accession number 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” means obtained and separated from a particular origin. The term “isolated” further means not being in its respective naturally occurring form, circumstances and / or environment. For example, “isolated from streptococcus” means a substance obtained and separated from streptococcal cells. Isolated polysaccharides are not naturally occurring. The term “isolated” means that the material has been removed from its original environment (e.g., from its natural environment if it is naturally occurring, or from its host organism if it is a recombinant entity, or transported from one environment to another). For example, an “isolated” capsular polysaccharide, protein, or peptide is substantially free of cellular material or other contaminating proteins from the cellular or tissue origin from which the protein is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals if present in a mixture as part of a chemical reaction. In the present invention, proteins or polysaccharides can be isolated from bacterial cells or cellular debris so as to be provided in a form useful in the production of immunogenic compositions. The terms “isolated” or “isolating” may include purification or cleansing, encompassing methods for purifying isolated polysaccharides that are known in the art and / or methods described herein. The term “substantially free of cellular material” encompasses polypeptide / protein preparations in which the polypeptide / protein has been separated from the cellular components of the cell that produced it by isolation or recombination. Thus, substantially free of cellular material, capsular polysaccharides, proteins or peptides encompass capsular polysaccharides, proteins or peptide preparations 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 produced by recombination, this preferably also substantially free of culture medium, i.e., the culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.When polypeptides / proteins or polysaccharides are produced by chemical synthesis, they preferably contain substantially no chemical precursors or other chemicals; that is, they are isolated from the chemical precursors or other chemicals involved in the synthesis of the protein or polysaccharide. Thus, such preparations of polypeptides / proteins or polysaccharides contain less than about 30%, 20%, 10%, or 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 present invention, the polysaccharide is isolated from bacteria. In another embodiment of the present invention, the polysaccharide is produced by recombinant DNA. In a further embodiment, the polysaccharide is synthesized or chemically synthesized according to conventional methods. In yet another embodiment of the present invention, the polysaccharide is prepared by expression in a surrogate host after cloning and expression of a biosynthetic pathway for producing the polysaccharide. In one embodiment, the polysaccharide is immunogenic. For example, the inventors have found that each polysaccharide described herein can induce or derive an immune response. The term “immunogenicity” refers to the ability to initiate, trigger, induce, enhance, improve, and / or enhance humoral and / or cell-mediated immune responses in mammals. In one embodiment, mammals include humans, primates, rabbits, pigs, mice, and the like.

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

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

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

[0134] In one embodiment, the polysaccharides described herein can induce opsonin activity. In another embodiment, the polysaccharides described herein can induce opsonin and phagocytic activity (e.g., opsonin-phagocytic activity).

[0135] 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 as described herein), which facilitates the binding of the antigen to phagocytic cells (e.g., macrophages, dendritic cells, and polymorphonuclear leukocytes (PMNL)). Some bacteria, such as encapsulated bacteria that are not typically phagocytosed due to the presence of a capsule, become more easily recognized by phagocytic cells when coated with an opsonin antibody. In one embodiment, the polysaccharide induces an immune response, such as an antibody, which is an opsonin. In one embodiment, the opsonization activity is against Gram-positive cocci, preferably against Streptococcus species, and more preferably against at least one strain of S. agalactiae.

[0136] In another embodiment, the polysaccharides described herein can induce a bactericidal immune response. In one embodiment, the bactericidal activity is against Gram-positive cocci, preferably against Streptococcus species, and 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 the reduction of bacterial load in vivo (e.g., by measuring the level of bacteremia in mammals inoculated with a species of Streptococcus) and / or by measuring bacterial cell death in vitro (e.g., an in vitro opsonization-phagocytosis assay). In one embodiment, a polysaccharide can induce opsonization, phagocytosis, and / or bactericidal activity compared to a suitable control, for example, compared to antiserum grown against heat-killed Gram-positive cocci.

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

[0139] [ka]

[0140] The molecular weight of serotype Ia capsular polysaccharides before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 25 kDa and approximately 750 kDa, between approximately 25 kDa and approximately 500 kDa, between approximately 25 kDa and approximately 450 kDa, between approximately 25 kDa and approximately 400 kDa, between approximately 25 kDa and approximately 350 kDa, between approximately 25 kDa and approximately 300 kDa, between approximately 25 kDa and approximately 250 kDa, between approximately 25 kDa and approximately 200 kDa, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, approximately Between 50kDa and approximately 400kDa, between approximately 50kDa and approximately 350kDa, between approximately 50kDa and approximately 300kDa, between approximately 50kDa and approximately 250kDa, between approximately 50kDa and approximately 200kDa, between approximately 75kDa and approximately 750kDa, between approximately 75kDa and approximately 500kDa, between approximately 75kDa and approximately 450kDa, between approximately 75kDa and approximately 400kDa, between approximately 75kDa and approximately 350kDa, between approximately 75kDa and approximately 300kDa, between approximately 75kDa and approximately 250kDa, between approximately 75kDa and approximately 200kDa, and between approximately 100kDa and approximately 750kDa Between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, between approximately 100kDa and 400kDa, between approximately 100kDa and 350kDa, between approximately 100kDa and 300kDa, between approximately 200kDa and 750kDa, between approximately 200kDa and 700kDa, between approximately 200kDa and 650kDa, between approximately 200kDa and 600kDa, Between approximately 200kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa and 700kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa,The molecular weights are approximately between 300 kDa and 700 kDa, between 300 kDa and 650 kDa, between 300 kDa and 600 kDa, between 300 kDa and 550 kDa, or between 300 kDa and 500 kDa, etc. In one preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 25 kDa and 200 kDa. In another preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 100 kDa and 400 kDa. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

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

[0142] In one embodiment, the serotype Ia capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0143] In another embodiment, the serotype Ia capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0144] Serotype Ia capsular polysaccharides are O-acetylated to less than approximately 5%. Some exemplary strains of serotype Ia capsular polysaccharides of the present invention include 090, A909 (ATCC accession number BAA-1138), 515 (ATCC accession number BAA-1177), B523, CJB524, and MB4052 (ATCC accession number 31574).

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

[0146] [ka]

[0147] The molecular weight of serotype Ib capsular polysaccharides before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 25 kDa and approximately 750 kDa, between approximately 25 kDa and approximately 500 kDa, between approximately 25 kDa and approximately 450 kDa, between approximately 25 kDa and approximately 400 kDa, between approximately 25 kDa and approximately 350 kDa, between approximately 25 kDa and approximately 300 kDa, between approximately 25 kDa and approximately 250 kDa, between approximately 25 kDa and approximately 200 kDa, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, Between 50kDa and approximately 400kDa, between approximately 50kDa and approximately 350kDa, between approximately 50kDa and approximately 300kDa, between approximately 50kDa and approximately 250kDa, between approximately 50kDa and approximately 200kDa, between approximately 75kDa and approximately 750kDa, between approximately 75kDa and approximately 500kDa, between approximately 75kDa and approximately 450kDa, between approximately 75kDa and approximately 400kDa, between approximately 75kDa and approximately 350kDa, between approximately 75kDa and approximately 300kDa, between approximately 75kDa and approximately 250kDa, between approximately 75kDa and approximately 200kDa, and between approximately 100kDa and approximately 750kDa Between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, between approximately 100kDa and 400kDa, between approximately 100kDa and 350kDa, between approximately 100kDa and 300kDa, between approximately 200kDa and 750kDa, between approximately 200kDa and 700kDa, between approximately 200kDa and 650kDa, between approximately 200kDa and 600kDa, Between approximately 200kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa and 700kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa,The molecular weights are approximately between 300 kDa and 700 kDa, between 300 kDa and 650 kDa, between 300 kDa and 600 kDa, between 300 kDa and 550 kDa, or between 300 kDa and 500 kDa, etc. In one preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 25 kDa and 400 kDa. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0148] In one embodiment, the serotype Ib capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0149] In another embodiment, the serotype Ib capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0150] Serotype Ib capsular polysaccharides are O-acetylated between approximately 0% and approximately 40%. In one embodiment of the present invention, the polysaccharide is O-deacetylated (i.e., O-acetylated to less than approximately 5%). Some exemplary strains of serotype Ib capsular polysaccharides of the present invention include H36B (ATCC accession number 12401), S40, S42, MB4053 (ATCC accession number 31575), M709, 133, 7357, and PFEGBST0267.

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

[0152] [ka]

[0153] The molecular weight of serotype II capsular polysaccharides before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 25 kDa and approximately 750 kDa, between approximately 25 kDa and approximately 500 kDa, between approximately 25 kDa and approximately 450 kDa, between approximately 25 kDa and approximately 400 kDa, between approximately 25 kDa and approximately 350 kDa, between approximately 25 kDa and approximately 300 kDa, between approximately 25 kDa and approximately 250 kDa, between approximately 25 kDa and approximately 200 kDa, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, approximately Between 50kDa and approximately 400kDa, between approximately 50kDa and approximately 350kDa, between approximately 50kDa and approximately 300kDa, between approximately 50kDa and approximately 250kDa, between approximately 50kDa and approximately 200kDa, between approximately 75kDa and approximately 750kDa, between approximately 75kDa and approximately 500kDa, between approximately 75kDa and approximately 450kDa, between approximately 75kDa and approximately 400kDa, between approximately 75kDa and approximately 350kDa, between approximately 75kDa and approximately 300kDa, between approximately 75kDa and approximately 250kDa, between approximately 75kDa and approximately 200kDa, and between approximately 100kDa and approximately 750kDa Between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, between approximately 100kDa and 400kDa, between approximately 100kDa and 350kDa, between approximately 100kDa and 300kDa, between approximately 200kDa and 750kDa, between approximately 200kDa and 700kDa, between approximately 200kDa and 650kDa, between approximately 200kDa and 600kDa, Between approximately 200kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa and 700kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa,The molecular weights are approximately between 300 kDa and 700 kDa, between 300 kDa and 650 kDa, between 300 kDa and 600 kDa, between 300 kDa and 550 kDa, or between 300 kDa and 500 kDa, etc. In one preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 25 kDa and 400 kDa. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0154] In one embodiment, the serotype II capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0155] In another embodiment, the serotype II capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0156] Serotype II capsular polysaccharides are O-acetylated to less than approximately 5%. Some exemplary strains of serotype II capsular polysaccharides of the present invention include MB4055 (ATCC accession number 31576), 18RS21 (ATCC accession number BAA-1175), S16, S20, V8 (ATCC accession number 12973), DK21, DK23, UAB, 5401, and PFEGBST0708.

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

[0158] [ka]

[0159] The molecular weight of the serotype II capsular polysaccharide before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 25 kDa and approximately 750 kDa, between approximately 25 kDa and approximately 500 kDa, between approximately 25 kDa and approximately 450 kDa, between approximately 25 kDa and approximately 400 kDa, between approximately 25 kDa and approximately 350 kDa, between approximately 25 kDa and approximately 300 kDa, between approximately 25 kDa and approximately 250 kDa, between approximately 25 kDa and approximately 200 kDa, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, approximately Between 50kDa and approximately 400kDa, between approximately 50kDa and approximately 350kDa, between approximately 50kDa and approximately 300kDa, between approximately 50kDa and approximately 250kDa, between approximately 50kDa and approximately 200kDa, between approximately 75kDa and approximately 750kDa, between approximately 75kDa and approximately 500kDa, between approximately 75kDa and approximately 450kDa, between approximately 75kDa and approximately 400kDa, between approximately 75kDa and approximately 350kDa, between approximately 75kDa and approximately 300kDa, between approximately 75kDa and approximately 250kDa, between approximately 75kDa and approximately 200kDa, and between approximately 100kDa and approximately 750kDa Between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, between approximately 100kDa and 400kDa, between approximately 100kDa and 350kDa, between approximately 100kDa and 300kDa, between approximately 200kDa and 750kDa, between approximately 200kDa and 700kDa, between approximately 200kDa and 650kDa, between approximately 200kDa and 600kDa, Between approximately 200kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa and 700kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa,The molecular weights are approximately between 300 kDa and 700 kDa, between 300 kDa and 650 kDa, between 300 kDa and 600 kDa, between 300 kDa and 550 kDa, or between 300 kDa and 500 kDa, etc. In one preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 25 kDa and 200 kDa. In another preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 100 kDa and 400 kDa. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

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

[0161] In one embodiment, the serotype III capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0162] In another embodiment, the serotype III capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0163] Serotype III capsular polysaccharides are O-acetylated between approximately 0% and approximately 40%. In one embodiment of the present invention, the polysaccharide is O-deacetylated (i.e., O-acetylated to less than approximately 5%). Some exemplary strains of serotype III capsular polysaccharides of the present invention include MB4082 (ATCC accession number 31577), M132, 110, M781 (ATCC accession number BAA-22), D136C(3) (ATCC accession number 12403), M782, S23, 120, MB4316 (M-732; ATCC accession number 31475), M132, K79, COH1 (ATCC accession number BAA-1176), and PFEGBST0563.

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

[0165] [ka]

[0166] The molecular weight of serotype IV capsular polysaccharides before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 25 kDa and approximately 750 kDa, between approximately 25 kDa and approximately 500 kDa, between approximately 25 kDa and approximately 450 kDa, between approximately 25 kDa and approximately 400 kDa, between approximately 25 kDa and approximately 350 kDa, between approximately 25 kDa and approximately 300 kDa, between approximately 25 kDa and approximately 250 kDa, between approximately 25 kDa and approximately 200 kDa, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, Between 50kDa and approximately 400kDa, between approximately 50kDa and approximately 350kDa, between approximately 50kDa and approximately 300kDa, between approximately 50kDa and approximately 250kDa, between approximately 50kDa and approximately 200kDa, between approximately 75kDa and approximately 750kDa, between approximately 75kDa and approximately 500kDa, between approximately 75kDa and approximately 450kDa, between approximately 75kDa and approximately 400kDa, between approximately 75kDa and approximately 350kDa, between approximately 75kDa and approximately 300kDa, between approximately 75kDa and approximately 250kDa, between approximately 75kDa and approximately 200kDa, and between approximately 100kDa and approximately 750kDa Between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, between approximately 100kDa and 400kDa, between approximately 100kDa and 350kDa, between approximately 100kDa and 300kDa, between approximately 200kDa and 750kDa, between approximately 200kDa and 700kDa, between approximately 200kDa and 650kDa, between approximately 200kDa and 600kDa, Between approximately 200kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa and 700kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa,The molecular weights are approximately between 300 kDa and 700 kDa, between 300 kDa and 650 kDa, between 300 kDa and 600 kDa, between 300 kDa and 550 kDa, or between 300 kDa and 500 kDa, etc. In one preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 25 kDa and 400 kDa. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0167] In one embodiment, the serotype IV capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0168] In another embodiment, the serotype IV capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0169] Serotype IV capsular polysaccharides are O-acetylated between approximately 0% and approximately 40%. In one embodiment of the present invention, the polysaccharide is O-deacetylated (i.e., O-acetylated to less than approximately 5%). Some exemplary strains of serotype IV capsular polysaccharides of the present invention include 3139 (ATCC accession number 49446), CZ-NI-016, and PFEGBST0961.

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

[0171] [ka]

[0172] The molecular weight of serotype V capsular polysaccharide before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 25 kDa and approximately 750 kDa, between approximately 25 kDa and approximately 500 kDa, between approximately 25 kDa and approximately 450 kDa, between approximately 25 kDa and approximately 400 kDa, between approximately 25 kDa and approximately 350 kDa, between approximately 25 kDa and approximately 300 kDa, between approximately 25 kDa and approximately 250 kDa, between approximately 25 kDa and approximately 200 kDa, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, and approximately 5 Between 0kDa and approximately 400kDa, between approximately 50kDa and approximately 350kDa, between approximately 50kDa and approximately 300kDa, between approximately 50kDa and approximately 250kDa, between approximately 50kDa and approximately 200kDa, between approximately 75kDa and approximately 750kDa, between approximately 75kDa and approximately 500kDa, between approximately 75kDa and approximately 450kDa, between approximately 75kDa and approximately 400kDa, between approximately 75kDa and approximately 350kDa, between approximately 75kDa and approximately 300kDa, between approximately 75kDa and approximately 250kDa, between approximately 75kDa and approximately 200kDa, and between approximately 100kDa and approximately 750kDa Between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, between approximately 100kDa and 400kDa, between approximately 100kDa and 350kDa, between approximately 100kDa and 300kDa, between approximately 200kDa and 750kDa, between approximately 200kDa and 700kDa, between approximately 200kDa and 650kDa, between approximately 200kDa and 600kDa, Between approximately 200kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa and 700kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa,The molecular weights are approximately between 300 kDa and 700 kDa, between 300 kDa and 650 kDa, between 300 kDa and 600 kDa, between 300 kDa and 550 kDa, or between 300 kDa and 500 kDa, etc. In one preferred embodiment, the molecular weight of the pre-conjugation capsular polysaccharide is between 25 kDa and 400 kDa. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0173] In one embodiment, the serotype V capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0174] In another embodiment, the serotype V capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0175] Serotype V capsular polysaccharides are O-acetylated between approximately 0% and approximately 40%. In one embodiment of the present invention, the polysaccharide is O-deacetylated (i.e., O-acetylated to less than approximately 5%). Some exemplary strains of serotype V capsular polysaccharides of the present invention include 1169-NT1, CJB111 (ATCC accession number BAA-23), CJB112, 2603V / R (ATCC accession number BAA-611), NCTC10 / 81, CJ11, and PFEGBST0837.

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

[0177] [ka]

[0178] The molecular weight of serotype VI capsular polysaccharides before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, between approximately 50 kDa and approximately 400 kDa, between approximately 50 kDa and approximately 350 kDa, between approximately 50 kDa and approximately 300 kDa, between approximately 50 kDa and approximately 250 kDa, between approximately 50 kDa and approximately 200 kDa, between approximately 75 kDa and approximately 750 kDa, between approximately 75 kDa and approximately 500 kDa, and between approximately 75 kDa and approximately Between 450kDa, between approximately 75kDa and 400kDa, between approximately 75kDa and 350kDa, between approximately 75kDa and 300kDa, between approximately 75kDa and 250kDa, between approximately 75kDa and 200kDa, between approximately 100kDa and 750kDa, between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, approximately 100kD Between a and approximately 400kDa, between approximately 100kDa and approximately 350kDa, between approximately 100kDa and approximately 300kDa, between approximately 200kDa and approximately 750kDa, between approximately 200kDa and approximately 700kDa, between approximately 200kDa and approximately 650kDa, between approximately 200kDa and approximately 600kDa, between approximately 200kDa and approximately 550kDa, between approximately 200kDa and approximately 500kDa, between approximately 200kDa and approximately 450kDa, between approximately 200kDa and approximately 400kDa, between approximately 250kDa and approximately 750kDa, between approximately 250kDa and approximately 700kDa The ranges are approximately 250kDa to 650kDa, 250kDa to 600kDa, 250kDa to 550kDa, 250kDa to 500kDa, 250kDa to 450kDa, 250kDa to 400kDa, 300kDa to 750kDa, 300kDa to 700kDa, 300kDa to 650kDa, 300kDa to 600kDa, 300kDa to 550kDa, or 300kDa to 500kDa, etc. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0179] In one embodiment, the serotype VI capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0180] In another embodiment, the serotype VI capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

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

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

[0183] [ka]

[0184] The molecular weight of serotype VII capsular polysaccharide before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, between approximately 50 kDa and approximately 400 kDa, between approximately 50 kDa and approximately 350 kDa, between approximately 50 kDa and approximately 300 kDa, between approximately 50 kDa and approximately 250 kDa, between approximately 50 kDa and approximately 200 kDa, between approximately 75 kDa and approximately 750 kDa, between approximately 75 kDa and approximately 500 kDa, and from approximately 75 kDa Between approximately 450kDa, between approximately 75kDa and 400kDa, between approximately 75kDa and 350kDa, between approximately 75kDa and 300kDa, between approximately 75kDa and 250kDa, between approximately 75kDa and 200kDa, between approximately 100kDa and 750kDa, between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, and approximately 100k Between Da and approximately 400kDa, between approximately 100kDa and approximately 350kDa, between approximately 100kDa and approximately 300kDa, between approximately 200kDa and approximately 750kDa, between approximately 200kDa and approximately 700kDa, between approximately 200kDa and approximately 650kDa, between approximately 200kDa and approximately 600kDa, between approximately 200kDa and approximately 550kDa, between approximately 200kDa and approximately 500kDa, between approximately 200kDa and approximately 450kDa, between approximately 200kDa and approximately 400kDa, between approximately 250kDa and approximately 750kDa, between approximately 250kDa and approximately 700kDa The ranges are as follows: between kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa, between approximately 300kDa and 700kDa, between approximately 300kDa and 650kDa, between approximately 300kDa and 600kDa, between approximately 300kDa and 550kDa, or between approximately 300kDa and 500kDa, etc. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0185] In one embodiment, the serotype VII capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0186] In another embodiment, the serotype VII capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0187] Serotype VII capsular polysaccharides are O-acetylated to less than approximately 5%. Exemplary strains of some serotype VII capsular polysaccharides of the present invention include 7271 and CZ-PW-045.

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

[0189] [ka]

[0190] The molecular weight of serotype VIII capsular polysaccharide before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, between approximately 50 kDa and approximately 400 kDa, between approximately 50 kDa and approximately 350 kDa, between approximately 50 kDa and approximately 300 kDa, between approximately 50 kDa and approximately 250 kDa, between approximately 50 kDa and approximately 200 kDa, between approximately 75 kDa and approximately 750 kDa, between approximately 75 kDa and approximately 500 kDa, and approximately 75 kDa. Between approximately 450kDa, between approximately 75kDa and 400kDa, between approximately 75kDa and 350kDa, between approximately 75kDa and 300kDa, between approximately 75kDa and 250kDa, between approximately 75kDa and 200kDa, between approximately 100kDa and 750kDa, between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, and approximately 100k Between Da and approximately 400kDa, between approximately 100kDa and approximately 350kDa, between approximately 100kDa and approximately 300kDa, between approximately 200kDa and approximately 750kDa, between approximately 200kDa and approximately 700kDa, between approximately 200kDa and approximately 650kDa, between approximately 200kDa and approximately 600kDa, between approximately 200kDa and approximately 550kDa, between approximately 200kDa and approximately 500kDa, between approximately 200kDa and approximately 450kDa, between approximately 200kDa and approximately 400kDa, between approximately 250kDa and approximately 750kDa, between approximately 250kDa and approximately 700kDa The ranges are as follows: between kDa, between approximately 250kDa and 650kDa, between approximately 250kDa and 600kDa, between approximately 250kDa and 550kDa, between approximately 250kDa and 500kDa, between approximately 250kDa and 450kDa, between approximately 250kDa and 400kDa, between approximately 300kDa and 750kDa, between approximately 300kDa and 700kDa, between approximately 300kDa and 650kDa, between approximately 300kDa and 600kDa, between approximately 300kDa and 550kDa, or between approximately 300kDa and 500kDa, etc. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0191] In one embodiment, the serotype VIII capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0192] In another embodiment, the serotype VIII capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

[0193] Serotype VIII capsular polysaccharides are O-acetylated between approximately 0% and approximately 40%. In one embodiment of the present invention, the polysaccharide is O-deacetylated (i.e., O-acetylated to less than approximately 5%). Exemplary strains of some of the serotype VIII capsular polysaccharides of the present 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 arrangement of GlcpNAc in the GBS serotype IX polysaccharide skeleton is alpha (α), which differs from previously published structural details where this binding was proposed to be in a β configuration. The structure of serotype IX can be more accurately described as follows:

[0195] [ka]

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

[0197] [ka]

[0198] The molecular weight of serotype IX capsular polysaccharide before conjugation is between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 50 kDa and approximately 750 kDa, between approximately 50 kDa and approximately 500 kDa, between approximately 50 kDa and approximately 450 kDa, between approximately 50 kDa and approximately 400 kDa, between approximately 50 kDa and approximately 350 kDa, between approximately 50 kDa and approximately 300 kDa, between approximately 50 kDa and approximately 250 kDa, between approximately 50 kDa and approximately 200 kDa, between approximately 75 kDa and approximately 750 kDa, between approximately 75 kDa and approximately 500 kDa, and between approximately 75 kDa and approximately Between 450kDa, between approximately 75kDa and 400kDa, between approximately 75kDa and 350kDa, between approximately 75kDa and 300kDa, between approximately 75kDa and 250kDa, between approximately 75kDa and 200kDa, between approximately 100kDa and 750kDa, between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, between approximately 100kDa and 450kDa, approximately 100kD Between a and approximately 400kDa, between approximately 100kDa and approximately 350kDa, between approximately 100kDa and approximately 300kDa, between approximately 200kDa and approximately 750kDa, between approximately 200kDa and approximately 700kDa, between approximately 200kDa and approximately 650kDa, between approximately 200kDa and approximately 600kDa, between approximately 200kDa and approximately 550kDa, between approximately 200kDa and approximately 500kDa, between approximately 200kDa and approximately 450kDa, between approximately 200kDa and approximately 400kDa, between approximately 250kDa and approximately 750kDa, between approximately 250kDa and approximately 700kDa The ranges are approximately 250kDa to 650kDa, 250kDa to 600kDa, 250kDa to 550kDa, 250kDa to 500kDa, 250kDa to 450kDa, 250kDa to 400kDa, 300kDa to 750kDa, 300kDa to 700kDa, 300kDa to 650kDa, 300kDa to 600kDa, 300kDa to 550kDa, or 300kDa to 500kDa, etc. Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0199] In one embodiment, the serotype IX capsular polysaccharide contains its natural sialic acid level, such as about 100% or more than about 95%. In another embodiment, the capsular polysaccharide may be desiallated before conjugation to a maximum of about 40% (sialylation level of more than about 60%), for example, up to about 35% (sialylation level of more than about 65%), up to about 30% (sialylation level of more than about 70%), up to about 25% (sialylation level of more than about 75%), up to about 20% (sialylation level of more than about 80%), up to about 15% (sialylation level of more than about 85%), up to about 10% (sialylation level of more than about 90%), or up to about 5% (sialylation level of more than about 95%).

[0200] In another embodiment, the serotype IX capsular polysaccharide has, prior to conjugation, about 1.0 mM sialic acid per mM polysaccharide, for example, at least about 0.95 mM sialic acid per mM polysaccharide. In yet another embodiment, the capsular polysaccharide may have, prior to conjugation, at least about 0.6 mM sialic acid per mM polysaccharide, for example, 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.

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

[0202] Polysaccharide-protein conjugate As used herein, “conjugate” typically comprises a capsular polysaccharide and a carrier protein of a desired molecular weight range, where the capsular polysaccharide is conjugated with the carrier protein. The conjugate may or may not contain small amounts 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 bonded to, adsorbed to, or captured in or by) the conjugated capsular polysaccharide-carrier protein. The terms “free capsular polysaccharide,” “free polysaccharide,” and “free sugar” may be used interchangeably and are intended to convey the same meaning. Regardless of the properties of the carrier molecule, the carrier molecule can conjugate with a capsular polysaccharide either directly or via a linker. As used herein, “conjugate,” “being conjugated,” and “conjugate” refer to the process by which a bacterial capsular polysaccharide covalently bonds with 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] When used herein, "conjugate immunogenic composition" refers to an immunogenic composition in which the immunogenic material comprises an antigenic polysaccharide that covalently binds to a carrier protein to provide a polysaccharide-protein conjugate. In one embodiment, the polysaccharide-protein conjugate of the present invention may be formulated as a polyvalent immunogenic composition.

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

[0205] As used herein, “polysaccharide-protein conjugate” refers to a polysaccharide molecule conjugated with a protein carrier molecule via one or more covalent bonds. It may be desirable to conjugate the polysaccharide with a protein from another species known to be immunogenic in a target host. Therefore, in one embodiment, the carrier molecule is a carrier protein. Such an exogenous protein is referred to as a “carrier protein” as defined herein. The carrier protein serves to enhance the antigenicity and immunogenicity of the polysaccharide. As used herein, the term “carrier effect” refers to the process by which the antigenicity and immunogenicity of a weakly immunogenic or non-immunogenic molecule are enhanced by conjugation 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 a T cell epitope to stimulate T cells that help produce an antibody response.

[0206] "Carrier protein" or "protein carrier," as used herein, refers to any protein molecule that can be conjugated with an antigen (such as a capsular polysaccharide) for which an immune response is desired. Conjugation of an antigen such as a polysaccharide with a carrier protein can make the antigen immunogenic. The carrier protein is preferably a protein that is non-toxic and non-reactive and can be obtained in sufficient quantity and purity. Examples of carrier proteins include toxins, toxoids, or any mutant cross-reactive material (CRM) of toxins derived from tetanus, diphtheria, pertussis, Pseudomonas species, Escherichia coli, Staphylococcus species, and Streptococcus species. 197 The carrier protein must be suitable for standard conjugation procedures. In one embodiment, the carrier protein is Streptococcus C5a peptidase (SCP). In another embodiment of the present invention, CRM 197 It is used as a carrier protein.

[0207] Cross-reactive material or CRM is particularly useful in some embodiments of the present invention. Genetically modified proteins can be generated that are antigenically similar but non-toxic to certain bacterial toxins. These are referred to as "cross-reactive materials" or CRM. CRM 197 (Wyeth / Pfizer Inc., Sanford, NC) is notable for having a single amino acid change from native diphtheria toxin and being immunologically indistinguishable therefrom. See Pappenheimer, A.M. et al., Immunochem., 9(9):891-906 (1972); U.S. Patent No. 5,614,382, the disclosure of which is incorporated herein by reference in its entirety. CRM 197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from a culture of Corynebacterium diphtheriae strain C7(β197) grown in a casamino acid and yeast extract-based medium. CRM 197 is purified via ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. CRM 197 The culture of Corynebacterium diphtheriae (C. diphtheriae) strain C7(β197) that produces the protein was deposited with the American Type Culture Collection, Rockville, Maryland, and was assigned the accession number ATCC 53281. Other diphtheria toxoids are also suitable for use as carrier proteins. CRM3201 is a genetically engineered variant of pertussis toxin. See Black, W.J. 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-fixed pathogenic 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 the recruitment of polymorphonuclear cells to the site of infection (2005.PNAS.102(51):18391.). It is a target of protective antibodies, and IgG antibodies directed at SCP can mediate opsonization phagocytosis. In addition, SCP can serve as a carrier protein that enhances the immune response to the conjugated GBS CPS polysaccharide hapten.

[0209] Diphtheria toxoid (DT), CRM 197In addition to SCP and pertussis toxoids, further examples of carrier proteins include tetanus toxoid (TT), cholera toxoid (as described, e.g., in International Patent Application Publication No. WO2004 / 083251), Escherichia coli (E. coli) heat-labile toxoid (LT), Escherichia coli (E. coli) heat-stable toxoid (ST), pneumococcal hemolysin (wild-type or reduced-toxicity mutant) derived from Streptococcus pneumoniae, pneumococcal surface protein A (PspA), pneumococcal adhesin protein A (PsaA), C5a peptidase derived from Streptococcus, hemolysin derived from Staphylococcus aureus, unclassifiable Haemophilus influenzae (NTHi) protein, Haemophilus influenzae protein D, and Clostridium perfringens. This includes Pseudomonas exotoxins / toxoids, hepatitis B surface antigens, hepatitis B core antigens, rotavirus VP7 protein, as well as respiratory syncytial virus F and G proteins, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derivatives of tuberculin (PPD), and Pseudomonas exotoxin or derivatives thereof of non-toxic mutant Pseudomonas aeruginosa exotoxin A produced by recombinant processes. Bacterial outer membrane proteins, such as outer membrane protein complex c (OMPC), porin, transferrin-binding protein, etc., or C. difficile enterotoxin (toxin A) and cytotoxin (toxin B) may also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivatives of tuberculin (PPD), can also be used as carrier proteins. In preferred embodiments, the carrier protein is diphtheria toxoid. More preferably, the carrier protein is CRM. 197 In another embodiment of the present invention, the carrier protein is tetanus toxoid.

[0210] For the synthesis of polyvalent conjugate immunogenic compositions, polysaccharide-protein conjugates can be produced by conjugating a mixture of polysaccharides purified from two different bacterial species with a carrier protein. Alternatively, polyvalent conjugate immunogenic compositions 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 may be mixed. Therefore, polyvalent immunogenic compositions may include carrier proteins carrying a homogeneous or heterogeneous population of conjugated polysaccharides.

[0211] Following 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. These techniques include, for example, concentration / hemodialysis filtration, precipitation / elution, column chromatography, and deep filtration.

[0212] As described above, the present invention relates to a conjugate comprising a GBS capsular polysaccharide conjugated with a carrier protein. One embodiment of the present invention provides a conjugate comprising a GBS serotype VI capsular polysaccharide conjugated with a carrier protein, and at least one additional conjugate comprising a GBS serotype Ia capsular polysaccharide conjugated with a carrier protein, a GBS serotype Ib capsular polysaccharide conjugated with a carrier protein, a GBS serotype II capsular polysaccharide conjugated with a carrier protein, a GBS serotype III capsular polysaccharide conjugated with a carrier protein, a GBS serotype V capsular polysaccharide conjugated with a carrier protein, a GBS serotype VII capsular polysaccharide conjugated with a carrier protein, a GBS serotype VIII capsular polysaccharide conjugated with a carrier protein, or a GBS serotype IX capsular polysaccharide conjugated with a carrier protein. In one embodiment of the present 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 20,000 kDa, and the conjugate contains less than 40% free polysaccharides compared to the total polysaccharide. In one embodiment, the conjugate contains less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% free polysaccharides compared to the total polysaccharide.

[0213] In one embodiment, serotype Ia, Ib, II, III, IV, V, VI, VII, VIII and / or IX polysaccharides are present in a range of about 5 kDa to about 1,000 kDa before conjugation, for example, 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, and between about 75 kDa and about 750 kDa. Between approximately 75kDa and 500kDa, between approximately 75kDa and 450kDa, between approximately 75kDa and 400kDa, between approximately 75kDa and 350kDa, between approximately 75kDa and 300kDa, between approximately 75kDa and 250kDa, between approximately 75kDa and 200kDa, between approximately 100kDa and 750kDa, between approximately 100kDa and 700kDa, between approximately 100kDa and 650kDa, between approximately 100kDa and 600kDa, between approximately 100kDa and 550kDa, between approximately 100kDa and 500kDa, and approximately 100kDa Between approximately 450kDa, between approximately 100kDa and 400kDa, between approximately 100kDa and 350kDa, between approximately 100kDa and 300kDa, between approximately 200kDa and 750kDa, between approximately 200kDa and 700kDa, between approximately 200kDa and 650kDa, between approximately 200kDa and 600kDa, between approximately 200kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa and 550kDa, between approximately 200kDa and 500kDa, between approximately 200kDa and 450kDa, between approximately 200kDa and 400kDa, between approximately 250kDa and 750kDa, between approximately 250kDa It has molecular weights ranging from approximately 700 kDa, from approximately 250 kDa to approximately 650 kDa, from approximately 250 kDa to approximately 600 kDa, from approximately 250 kDa to approximately 550 kDa, from approximately 250 kDa to approximately 500 kDa, from approximately 250 kDa to approximately 450 kDa, from approximately 250 kDa to approximately 400 kDa, from approximately 300 kDa to approximately 750 kDa, from approximately 300 kDa to approximately 700 kDa, from approximately 300 kDa to approximately 650 kDa, from approximately 300 kDa to approximately 600 kDa, from approximately 300 kDa to approximately 550 kDa, or from approximately 300 kDa to approximately 500 kDa.Any integer within any of the above ranges is intended as an embodiment of the present disclosure.

[0214] In one embodiment, the conjugate is between approximately 300 kDa and approximately 20,000 kDa, for example, between approximately 300 kDa and approximately 15,000 kDa, between approximately 300 kDa and approximately 10,000 kDa, between approximately 300 kDa and approximately 9,000 kDa, between approximately 300 kDa and approximately 8,000 kDa, between approximately 300 kDa and approximately 7,000 kDa, between approximately 300 kDa and approximately 6,000 kDa, between approximately 300 kDa and approximately 5,000 kDa, between approximately 300 kDa and approximately 4,000 kDa, between approximately 300 kDa and approximately 3,000 kDa, between approximately 300 kDa and approximately 2, Between 000kDa, between approximately 300kDa and 1,000kDa, between approximately 500kDa and 20,000kDa, between approximately 500kDa and 15,000kDa, between approximately 500kDa and 10,000kDa, between approximately 500kDa and 9,000kDa, between approximately 500kDa and 8,000kDa, between approximately 500kDa and 7,000kDa, between approximately 500kDa and 6,000kDa, between approximately 500kDa and 5,000kDa, between approximately 500kDa and 4,000kDa, between approximately 500kDa and 3,000kDa, and approximately 500k Between Da and approximately 2,000 kDa, between approximately 500 kDa and approximately 1,000 kDa, between approximately 1,000 kDa and approximately 20,000 kDa, between approximately 1,000 kDa and approximately 15,000 kDa, between approximately 1,000 kDa and approximately 10,000 kDa, between approximately 1,000 kDa and approximately 9,000 kDa, between approximately 1,000 kDa and approximately 8,000 kDa, between approximately 1,000 kDa and approximately 7,000 kDa, between approximately 1,000 kDa and approximately 6,000 kDa, between approximately 1,000 kDa and approximately 5,000 kDa, between approximately 1,500 kDa and approximately 20,000 kDa, Between approximately 1,500kDa and 15,000kDa, between approximately 1,500kDa and 10,000kDa, between approximately 1,500kDa and 9,000kDa, between approximately 1,500kDa and 8,000kDa, between approximately 1,500kDa and 7,000kDa, between approximately 1,500kDa and 6,000kDa, between approximately 1,500kDa and 5,000kDa, between approximately 2,000kDa and 20,000kDa, between approximately 2,000kDa and 15,000kDa, between approximately 2,000kDa and 10,000kDa, between approximately 2,000kDa and 9,Between 000kDa, between approximately 2,000kDa and 8,000kDa, between approximately 2,000kDa and 7,000kDa, between approximately 2,000kDa and 6,000kDa, between approximately 2,500kDa and 20,000kDa, between approximately 2,500kDa and 15,000kDa, between approximately 2,500kDa and 10,000kDa, between approximately 2,500kDa and 9,000kDa, between approximately 2,500kDa and 8,000kDa, and between approximately 2,500kDa and 7 It has molecular weights such as between 1,000 kDa, between approximately 2,500 kDa and 6,000 kDa, between approximately 3,000 kDa and 20,000 kDa, between approximately 3,000 kDa and 15,000 kDa, between approximately 3,000 kDa and 10,000 kDa, between approximately 3,000 kDa and 9,000 kDa, between approximately 3,000 kDa and 8,000 kDa, between approximately 3,000 kDa and 7,000 kDa, or between approximately 3,000 kDa and 6,000 kDa.

[0215] In one embodiment, the GBS serotype VI capsular polysaccharide conjugate has a molecular weight within the above range.

[0216] In one embodiment, the GBS serotype Ia capsular polysaccharide conjugate has a molecular weight within the above range.

[0217] In one embodiment, the GBS serotype Ib capsular polysaccharide conjugate has a molecular weight within the above range.

[0218] In one embodiment, the GBS serotype II capsular polysaccharide conjugate has a molecular weight within the above range.

[0219] In one embodiment, the GBS serotype III capsular polysaccharide conjugate has a molecular weight within the above range.

[0220] In one embodiment, the GBS serotype V capsular polysaccharide conjugate has a molecular weight within the above range.

[0221] In one embodiment, the GBS serotype VII capsular polysaccharide conjugate has a molecular weight within the above range.

[0222] In one embodiment, the GBS serotype VIII capsular polysaccharide conjugate has a molecular weight within the above range.

[0223] In one embodiment, the GBS serotype IX capsular polysaccharide conjugate has a molecular weight within the above range.

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

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

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

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

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

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

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

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

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

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

[0234] In one embodiment, the conjugate of the present invention contains less than 0.01, 0.02, 0.03, 0.04, or 0.05 mM of O-acetate per mM of sugar repeating units. In another embodiment, the conjugate contains at least 0.1, 0.2, 0.3, 0.35, or about 0.4 mM of O-acetate per mM of sugar repeating units.

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

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

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

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

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

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

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

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

[0243] In one embodiment, 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 contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of free GBS capsular polysaccharides, relative to the total amount of GBS capsular polysaccharides. In preferred embodiments, the immunogenic conjugate contains less than 5% of unreacted free sugars, relative to the total amount of GBS capsular polysaccharides.

[0245] In another embodiment, the GBS capsule polysaccharide to carrier protein ratio (weight / weight) in the conjugate is between about 0.5 and about 3.0. In one embodiment, the GBS capsule polysaccharide to carrier protein ratio 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 GBS capsule polysaccharide to carrier protein ratio 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, in which atoms from a polysaccharide are covalently bonded to atoms from the protein surface. Alternatively, conjugation can be mediated by linker molecules, which react with both the polysaccharide and the protein, linking the two together and attaching the carbohydrate to the protein.

[0248] When a carrier and one or more antigens, such as polysaccharides, are conjugated (i.e., covalently associated), the conjugation may be by any chemical method, process or genetic technique known to those skilled in the art. For example, a 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, may be (1) directly coupled via 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), or (3) homobifunctional coupling of thiols (e.g., using bis-maleimide). Conjugation may occur by techniques including, but not limited to, (1) using, (2) homobifunctional coupling via photoactivating reagents, (3) heterobifunctional coupling of amines with thiols (e.g., using maleimide), (4) heterobifunctional coupling via photoactivating reagents (e.g., the β-carbonyidiazo family), (5) introducing amine-reactive groups into polysaccharides or oligosaccharides via cyanide bromide activation or carboxymethylation, (6) introducing thiol-reactive groups into polysaccharides or oligosaccharides via heterobifunctional compounds such as maleimide-hydrazide, (7) protein-lipid conjugation via introducing hydrophobic groups into proteins, and (8) protein-lipid conjugation via incorporating reactive groups into lipids. Heterobifunctional "non-covalent coupling" techniques such as biotin-avidin interactions are also being considered. Other methods known in the art for achieving conjugation of oligosaccharides and polysaccharides with immunogenic carrier proteins are also within the scope of some embodiments of the present invention.

[0249] In one embodiment, the GBS capsule 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 the carrier protein, either directly or via a spacer (linker) group. For example, the spacer may be cystamine or cysteamine to obtain a thiolated polysaccharide that can be coupled to the carrier via a thioether bond obtained after a 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 prepared by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivative sugar is conjugated with the carrier protein using carbodiimide (e.g., EDAC or EDC) chemistry via the carboxyl group on the protein carrier. Such conjugates are described, for example, in International Patent Application Publications WO93 / 15760, WO95 / 08348, and WO96 / 29094.

[0251] Other preferred techniques use carbodiimides, hydrazides, active esters, norboranes, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication WO98 / 42721. The conjugation may involve the 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 a carbonyl linker which may be formed by reaction with a 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 of the CDI / CDT carbamate intermediate with an amino group on the protein.

[0252] In a preferred embodiment, the GBS capsule polysaccharide-protein conjugate of the present invention is prepared using reductive amination. Reductive amination involves two steps: (1) oxidation of the polysaccharide to produce an aldehyde functional group from an adjacent diol in each hexasaccharide unit, and (2) reduction of the activated polysaccharide and carrier protein to form a conjugate.

[0253] In one embodiment, GBS capsule polysaccharide is (a) A step of reacting isolated GBS capsule polysaccharide with an oxidizing agent, (b) A step in which the oxidation reaction product is quenched by the addition of a quenching agent to obtain activated GBS capsule polysaccharide. It is activated (oxidized) by a process that includes it.

[0254] In one embodiment of the present invention, the concentration of the isolated capsular polysaccharide is between about 0.1 mg / mL and about 10.0 mg / mL, for example, 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 a periodate. Periodates oxidize adjacent hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of the CC bond. The term "periodate" encompasses both periodates and periodic acids. This term also includes metaperiodates (IO4). - ) and orthoperiodate (IO6 5- This includes both of the above. The term "periodate" also includes various salts of periodates, including sodium periodate and potassium periodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In a preferred embodiment, the periodate used for the oxidation of GBS capsular polysaccharides is metaperiodate. In a preferred embodiment, the periodate used for the oxidation of serum-type capsular polysaccharides 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 an oxidizing agent. In a particular embodiment, 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 an oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.1 molar equivalents of an oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.15 molar equivalents of an oxidizing agent. In an additional embodiment, the polysaccharide is reacted with about 0.25 molar equivalents of an oxidizing agent. In yet another embodiment, the polysaccharide is reacted with about 0.5 molar equivalents of an oxidizing agent. In an alternative embodiment, the polysaccharide is reacted with about 0.6 molar equivalents of an oxidizing agent. In yet another embodiment, the polysaccharide is reacted with about 0.7 molar equivalents of an oxidizing agent.

[0257] In one embodiment of the present 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 embodiment of the present 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 reaction temperature is maintained at about 23°C. In another preferred embodiment, the reaction temperature is maintained at about 5°C.

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

[0260] In an additional aspect, the buffer has a concentration 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, for example, 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 capsule polysaccharide has a molecular weight between approximately 5 kDa and approximately 1,000 kDa, for example, between approximately 50 kDa and approximately 300 kDa, between approximately 75 kDa and approximately 400 kDa, between approximately 75 kDa and approximately 200 kDa, between approximately 100 kDa and approximately 700 kDa, between approximately 100 kDa and approximately 500 kDa, between approximately 100 kDa and approximately 400 kDa, between approximately 100 kDa and approximately 300 kDa, between approximately 200 kDa and approximately 400 kDa, and between approximately 300 kDa and approximately 700 kDa. In a preferred embodiment, the activated GBS capsule polysaccharide has a molecular weight between approximately 75 kDa and approximately 400 kDa.

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

[0268] In another embodiment, the activated GBS capsule polysaccharide is compounded with a carrier protein and freeze-dried, optionally in the presence of a sugar. In one embodiment, the sugar is selected from sucrose, trehalose, raffinose, stachyose, melegitose, dextran, mannitol, lactitol, and palatinite. In a preferred embodiment, the sugar is sucrose. The co-freeze-dried polysaccharide and carrier protein can then be resuspended in solution and reacted with a reducing agent.

[0269] Activated GBS capsule polysaccharide is (a) A step of combining activated GBS capsule polysaccharide with a carrier protein, (b) The step of reacting the formulated activated GBS capsule polysaccharide and carrier protein with a reducing agent to form a GBS capsule polysaccharide-carrier protein conjugate. It can be conjugated with a carrier protein by a process that includes [a specific component].

[0270] Conjugation of activated GBS capsule polysaccharides with protein carriers by reductive amination in a polar aprotic solvent is preferable for maintaining low levels of free polysaccharides compared to reductive amination in aqueous solution, for example, where the level of unreacted (free) polysaccharides 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 freeze-dried GBS capsule polysaccharide in a solution containing a carrier protein and a polar aprotic solvent. In another embodiment, step (a) comprises dissolving the co-freeze-dried GBS capsule polysaccharide and carrier protein in a polar aprotic solvent.

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

[0273] If steps (a) and (b) are carried out in an aqueous solution, steps (a) and (b) are preferably carried out in a buffer in an aqueous medium selected from PBS, MES, HEPES, Bis-Tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bisin, or HEPB, with 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. 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 approximately 0.1 mg / mL and approximately 10.0 mg / mL, between approximately 0.5 mg / mL and approximately 5.0 mg / mL, or between approximately 0.5 mg / mL and approximately 2.0 mg / mL. In a preferred embodiment, the concentration of activated serotype GBS capsular polysaccharide in step (b) is approximately 0.1 mg / mL, approximately 0.2 mg / mL, approximately 0.3 mg / mL, approximately 0.4 mg / mL, approximately 0.5 mg / mL, approximately 0.6 mg / mL, approximately 0.7 mg / mL, approximately 0.8 mg / mL, approximately 0.9 mg / mL, approximately 1.0 mg / mL, approximately 1.1 mg / mL, approximately 1.2 mg / mL, approximately 1.3 mg / mL, approximately 1 These are approximately 0.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, or 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 to 0.1:1, 2:1 to 0.1:1, 2:1 to 1:1, 1.5:1 to 1:1, 0.1:1 to 1:1, 0.3:1 to 1:1, and 0.6:1 to 1:1. In a preferred embodiment, the initial ratio of activated serotype GBS capsular polysaccharide to carrier protein is approximately 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, and 2:1.

[0276] In one embodiment, the reducing agent is a Brønsted or Lewis acid, an amine borane, such as pyridineborane, 2-picolinborane, 2,6-diborane-methanol, dimethylamine-borane, or 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-methylpyridineborane (PEMB), etc. In preferred embodiments, 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 reaction temperature 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 preferred embodiments, the reaction temperature in step (b) is maintained at approximately 23°C.

[0280] In an additional embodiment, a process for preparing an immunogenic conjugate comprising a GBS capsule polysaccharide covalently bound to a carrier protein further comprises a step (step (c)) of capping (quenching) unreacted aldehydes by adding boron hydride.

[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 hours and 10 hours, between 0.5 hours and 5 hours, or 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 of the GBS capsular polysaccharide with the carrier protein and capping, the polysaccharide-protein conjugate can be purified by various techniques known to those skilled in the art (enriched with respect to the amount of polysaccharide-protein conjugate). These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration, precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration.

[0287] In further embodiments, the immunogenic conjugate contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% of free GBS capsular polysaccharides, relative to the total amount of GBS capsular polysaccharides. In preferred embodiments, the immunogenic conjugate contains less than 5% of unreacted free sugars, relative to the total amount of GBS capsular polysaccharides.

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

[0289] In another embodiment, the GBS capsule polysaccharide to carrier protein ratio (weight / weight) in the conjugate is between about 0.5 and about 3.0. In one embodiment, the GBS capsule polysaccharide to carrier protein ratio 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 GBS capsule polysaccharide to carrier protein ratio 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 capsule polysaccharide-protein conjugate is obtained by the reductive amination method described above. For example, in one embodiment, the present disclosure is: (a) A step of reacting isolated GBS capsule polysaccharide with an oxidizing agent, (b) A step of quenching the oxidation reaction product by adding a quenching agent to obtain activated GBS capsule polysaccharide, (c) A step of combining activated GBS capsule polysaccharide with a carrier protein, (d) The step of reacting the formulated activated GBS capsule polysaccharide and carrier protein with a reducing agent to form a GBS capsule polysaccharide-carrier protein conjugate, and optionally (e) A step of capping unreacted aldehydes by adding sodium borohydride (NaBH4) The present invention provides a GBS capsule polysaccharide-protein conjugate containing a polysaccharide conjugated with a carrier protein, which can be produced or obtained by a method including the present invention.

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

[0293] In another embodiment of the present invention, the GBS capsule polysaccharide-protein conjugate of the present invention is 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 WO2014 / 097099, which is incorporated herein by reference in its entirety. In such embodiments, the glycoconjugate derived from GBS capsule polysaccharide is prepared using a TEMPO free radical to oxidize the primary alcohol of the sugar to an aldehyde (hereafter, "TEMPO / NCS oxidation") using NCS as a co-oxidant, as described in Example 7 and International Patent Application Publication WO2014 / 097099. Therefore, in one embodiment, a conjugate of GBS capsule polysaccharide can be obtained by a method comprising the steps of a) reacting GBS capsule polysaccharide with TEMPO and NCS in a solvent to produce an activated sugar, and b) reacting the activated sugar with a carrier protein containing one or more amine groups (hereinafter referred to as "TEMPO / NCS-reductive amination"). In one embodiment, the solvent may be an aqueous solvent or DMSO.

[0294] In one embodiment, the GBS capsule polysaccharide-protein conjugate is obtained by the method described above. For example, in one embodiment, the Disclosure provides a GBS capsule polysaccharide-protein conjugate comprising a polysaccharide conjugated with a carrier protein, which can be produced or obtained by a method comprising: a) reacting a sugar with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in a solvent to produce an activated sugar; and b) reacting the activated sugar 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 purifying the individual conjugates, they can be combined to formulate an immunogenic composition of the present invention, which may be used, for example, in a vaccine. The formulation of the immunogenic composition of the present invention can be carried out using methods accepted in the art.

[0296] An “immune response” to an immunogenic composition is the development of humoral and / or cell-mediated immune responses to molecules (e.g., antigens such as proteins or polysaccharides) present in the composition of interest. For the purposes of this invention, a “humoral immune response” is an antibody-mediated immune response, involving the production of antibodies with affinity for antigens present in the immunogenic composition of this invention, while a “cell-mediated immune response” is mediated by T lymphocytes and / or other leukocytes. A “cell-mediated immune response” is derived from the presentation of antigenic epitopes associated with class I or class II molecules of the major histocompatibility complex (MHC). This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic T lymphocytes (CTLs). CTLs are specific to peptide or lipid antigens that are presented in association with proteins encoded by MHC or CD1 and expressed on the cell surface. CTLs help induce and promote intracellular disruption of intracellular microorganisms, or 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 on the activity of nonspecific effector cells against cells displaying peptide antigens associated 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 other leukocytes, including those derived from activated T cells and / or 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 assaying T-lymphocytes specific to the antigen in a sensitized subject, for example, by lymphocyte proliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, etc., or by measuring cytokine production by T cells in response to restimulation with the antigen. Such assays are well known in the art.For example, see Erickson, AL 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, cell-mediated, or both immune response.

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

[0299] When used herein, “immune interference” or “significant immune interference” refers to a statistically significant reduction in the immune response to individual antigens in a multivalent or multicomponent vaccine compared to the immune response to the same antigen when administered with a monovalent vaccine.

[0300] A “protective” immune response refers to the ability of an immunogenic composition to induce either a humoral or cell-mediated immune response that helps protect a subject from infection. The protection provided does not need to be absolute; that is, it does not need to completely prevent or eradicate infection if there is a statistically significant improvement compared to a control population of the subject, e.g., infected animals that have not been administered either vaccine or immunogenic composition. Protection may be limited to mitigating the severity or speed of the onset of symptoms of infection. Several assays for determining whether an immune response exhibits a “protective immune response” are known in the art. For example, an increase in antibody levels can be measured by binding assays such as the whole-cell ELISA assay described later. Other assays include measuring functional antibody responses, such as facilitating bacterial killing, which can be tested by opsonin phagocytosis assays (OPAs) as described later. In certain circumstances, a “protective immune response” may include inducing a twofold or fourfold increase in antibody levels specific to a particular antigen in at least 50% of the subjects. In other contexts, a “protective immune response” may encompass a reduction in bacterial count 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 amount of conjugated and unconjugated polysaccharides for that conjugate. For example, a GBS capsular polysaccharide conjugate with 20% free polysaccharides is expected to contain approximately 80 mcg / ml of conjugated GBS capsular polysaccharide and approximately 20 mcg / ml of unconjugated GBS capsular polysaccharide in a 100 mcg / ml dose of GBS capsular polysaccharide. The contribution of protein carriers to the conjugate is usually not considered 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 approximately 0.01 mg / ml to approximately 100 mcg / ml of each polysaccharide, particularly approximately 1 mcg / ml to approximately 70 mcg / ml, and more preferably approximately 5 mcg / ml to approximately 50 mcg / ml. The "immunogenicity" of different polysaccharide components in an immunogenic composition may vary, and each may be approximately 0.01 mcg / ml, 0.1 mcg / ml, 0.25 mcg / ml, 0.5 mcg / ml, 1 mcg / ml, 2 mcg / ml, 3 mcg / ml, 4 mcg / ml, 5 mcg / ml, 6 mcg / ml, 7 mcg / ml, and 8 mcg / ml, respectively. It may contain any specific polysaccharide antigen in doses of mcg / ml, approximately 9mcg / ml, approximately 10mcg / ml, approximately 15mcg / ml, approximately 20mcg / ml, approximately 25mcg / ml, approximately 30mcg / ml, approximately 40mcg / ml, approximately 50mcg / ml, approximately 60mcg / ml, approximately 70mcg / ml, approximately 80mcg / ml, approximately 90mcg / ml, or approximately 100mcg / ml. The dose or immunogenicity of a polyvalent 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 efficacy of an antigen as an immunogen can be measured by measuring the level of circulating antibodies specific to the antigen in serum using immunoassays, immunoprecipitation assays, functional antibody assays such as in vitro opsonin assays, and many other assays known in the art, or by measuring the level of B cell activity. Another measurement of the efficacy of an antigen as a T cell immunogen can be measured by proliferation assays or by cell lysis assays such as chromium release assays to measure the ability of T cells to lyse their specific target cells. Furthermore, in this invention, “immunogenicity” may be defined by measuring the serum level of antigen-specific antibodies induced after administration of the antigen, or by measuring the ability of the antibodies thus induced to enhance the opsonization ability of specific 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 for which the immune response is desired is a bacterium, the level of protection induced by the “immunogenicity” of the antigen can be measured by detecting the survival percentage or mortality percentage after inoculation of the animal with bacterial cells. In one embodiment, the amount of protection may be measured by measuring at least one symptom associated with a bacterial infection, such as fever associated with the infection. The amount of each antigen in a multi-antigen or multi-component vaccine or immunogenic composition will vary with respect to each of the other components and can be determined by methods known to those skilled in the art. Such methods are expected to include, for example, procedures for measuring immunogenicity and / or in vivo efficacy.

[0303] The term “immunogenic composition” refers to any pharmaceutical composition containing an antigen, such as a microorganism, or a component thereof, which can be used to induce an immune response in a subject. The immunogenic compositions of the present invention can be used to treat humans susceptible to GBS infection by utilizing the administration of the immunogenic composition via systemic transdermal or mucosal routes. These administrations may include injection via intramuscular (im), intraperitoneal (ip), intradermal (id), or subcutaneous routes, application by patch or other transdermal delivery device, or via oral / digestive, respiratory, or mucosal administration to the urogenital tract. In one embodiment, the immunogenic composition may be used in the manufacture of vaccines or in the derivation of polyclonal or monoclonal antibodies that may be used to passively protect or treat animals.

[0304] In one embodiment, the present invention relates to an immunogenic composition comprising an effective amount of at least one polysaccharide, oligosaccharide, polysaccharide-protein conjugate, or bioequivalent thereof, as described herein. For example, in one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the capsular polysaccharide being selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX of Streptococcus group B, and the capsular polysaccharide having a sialic acid level greater than about 60%. In another example, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising a capsular polysaccharide derived from serotype VI of Streptococcus group B 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 derived from at least two additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII, and IX of Streptococcus group B. In yet another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides derived from at least three additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII, and IX of Streptococcus group B. In a further embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides derived from at least four additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VII, VIII, and IX of Streptococcus group B. In certain embodiments, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides derived from serotypes VI, VII, VIII, and IX of Group B Streptococcus. In other embodiments, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides derived from serotypes Ia, Ib, II, III, and VI of Group B Streptococcus.In another embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate, the conjugate comprising capsular polysaccharides derived from serotype VI of Group B Streptococcus 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, the conjugate comprising capsular polysaccharides derived from serotypes Ia, Ib, II, III, IV, and VI of Group B Streptococcus.

[0305] In one embodiment, the immunogenic composition of the present invention comprises 1 to 10 different serotypes of S. agalactiae. Thus, in one embodiment, the immunogenic composition of the present 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] Accordingly, the present invention relates to monovalent and / or polyvalent immunogenic compositions comprising a polysaccharide-protein conjugate 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 particular embodiment, the immunogenic composition comprises GBS capsular polysaccharide serotype VI.

[0307] A polysaccharide-protein conjugate may contain the same or different protein carriers. In one embodiment, the conjugate contains the same protein carrier, and the sugar is conjugated with the same molecule of the protein carrier (a carrier molecule having two or more different polysaccharides conjugated with it). See, for example, International Patent Application Publication WO2004 / 083251. In another embodiment, one or more polysaccharides are independently conjugated with different molecules of the protein carrier (each molecule of the protein carrier having only one type of polysaccharide conjugated with it). In such embodiments, the capsular sugar is said to be conjugated independently of the carrier protein.

[0308] The optimal amounts of components in a particular immunogenic composition can be determined through standard studies involving observation of the appropriate immune response in the subjects. After initial vaccination, subjects can receive one or more booster immunizations at appropriate intervals.

[0309] The immunogenic compositions of the present invention may further contain one or more preservatives in addition to a plurality of capsular polysaccharide protein conjugates. The FDA requires that biological products in multi-dose vials contain preservatives, with few exceptions. The present invention is intended for use in such multi-dose vials. Vaccine products containing preservatives include vaccines containing benzethonium chloride (anthrax), 2-phenoxyethanol (DTaP, HepA, lime, polio (parenteral)), and phenol (pneumonia, typhoid (parenteral)). Preservatives approved for use in injectable drugs include, for example, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, and phenylmercury nitrate.

[0310] In another embodiment, the present invention relates to compositions comprising at least one of any polysaccharides described herein, and pharmaceutically acceptable excipients, buffers, stabilizers, adjuvants, antifreeze agents, salts, divalent cations, nonionic surfactants, free radical oxidation inhibitors, diluents or carriers, or mixtures 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 preferred embodiments, the buffer is histidine.

[0312] In one embodiment, the immunogenic composition contains a buffer at concentrations ranging from 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 contains 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 contains histidine at a concentration of about 20 mM.

[0314] In a particular embodiment, the formulation is buffered within a pH range of about 5.0 to about 7.1, for example, 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 within a pH range of about 6.0 to about 7.0, most preferably about 6.5.

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

[0316] In one embodiment, the immunogenic composition contains surfactants in amounts of approximately 0.001% to approximately 2% (v / w), approximately 0.001% to approximately 1%, approximately 0.001% to approximately 0.5%, approximately 0.001% to approximately 0.1%, approximately 0.001% to approximately 0.05%, approximately 0.001% to approximately 0.01%, approximately 0.001% to approximately 0.005%, approximately 0.005% to approximately 2%, and approximately 0.005% Approximately 1%, approximately 0.005% to approximately 0.5%, approximately 0.005% to approximately 0.1%, approximately 0.005% to approximately 0.05%, approximately 0.005% to approximately 0.01%, approximately 0.01% to approximately 2%, approximately 0.01% to approximately 1%, approximately 0.01% to approximately 0.5%, approximately 0.01% to approximately 0.1%, approximately 0.01% to approximately 0.05%, approximately 0.01% to approximately 0.04%, approximately 0.01% to approximately 0 0.03%, approximately 0.015% to approximately 2%, approximately 0.015% to approximately 1%, approximately 0.015% to approximately 0.5%, approximately 0.015% to approximately 0.1%, approximately 0.015% to approximately 0.05%, approximately 0.015% to approximately 0.04%, approximately 0.015% to approximately 0.03%, approximately 0.02% to approximately 2%, approximately 0.02% to approximately 1%, approximately 0.02% to approximately 0.5%, approximately 0.02% to approximately 0. The immunogenic composition contains the surfactant in concentrations of 1%, 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 about 0.25%. In a preferred embodiment, the immunogenic composition contains the surfactant in 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) in a concentration of about 0.001% to about 2% (preferably up to about 0.25%) or polysorbate-40 in a concentration of about 0.001% to 1% (preferably up to about 0.5%).

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

[0319] A pharmaceutically acceptable carrier is used to bind the carbohydrate of the present invention to a protein and should not be confused with a “carrier protein” that modifies the immune response to that carbohydrate. To avoid confusion with protein carriers as described herein, the term pharmaceutically acceptable diluent is preferred over pharmaceutically acceptable carrier, although these terms may occasionally be used interchangeably. The term “pharmaceutically acceptable carrier” means a carrier approved by a federal regulatory agency, a state government or other regulatory agency, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, including human and non-human mammals. The term “carrier” refers to a diluent, adjuvant, excipient or vehicle with which the pharmaceutical composition is administered. Suitable pharmaceutically acceptable diluents include all kinds of conventional solvents, dispersions, fillers, solid carriers, aqueous solutions, coatings, antimicrobial and antifungal agents, isotonic and absorption retarders, etc. Such pharmaceutically acceptable diluents may be sterile liquids, e.g., water, and oils, including petroleum, animal, plant or synthetic origins. Water, water for injection (WFI), sterile isotonic saline, phosphate buffer solution, adjuvant suspension, aqueous dextrose and glycerol solutions, and combinations thereof can be used as liquid carriers, particularly for injection solutions. A pharmaceutically acceptable diluent may further contain small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, that enhance the shelf life or efficacy in the body. The preparation and use of pharmaceutically acceptable diluents are well known in the art. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. In one embodiment, the diluent is water, water for injection (WFI), adjuvant suspension, or saline. In a particular embodiment, the diluent is a suspension of any adjuvant 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, melegitose, dextran, mannitol, lactitol, palatinite, gelatin, malt, rice, wheat flour, stone powder, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. In a preferred embodiment, the excipient is NaCl.

[0321] In one embodiment, the immunogenic composition contains excipients in concentrations of approximately 10 mM to 500 mM, approximately 10 mM to 450 mM, approximately 10 mM to 400 mM, approximately 10 mM to 350 mM, approximately 10 mM to 300 mM, approximately 10 mM to 250 mM, approximately 10 mM to 200 mM, approximately 10 mM to 150 mM, approximately 10 mM to 100 mM, approximately 10 mM to 50 mM, approximately 10 mM to 30 mM, approximately 10 mM to 20 mM, 20 mM to 500 mM, approximately 20 mM to 450 mM, approximately 20 mM to 400 mM, and approximately 20 mM to 350 mM. Approximately 20mm to 300mm, approximately 20mm to 250mm, approximately 20mm to 200mm, approximately 20mm to 150mm, approximately 20mm to 100mm, approximately 20mm to 50mm, approximately 20mm to 30mm, approximately 50mm to 500mm, approximately 50mm to 450mm, approximately 50mm to 400mm, approximately 50mm to 350mm, approximately 50mm to 300mm, approximately 50mm to 250mm, approximately 50mm to 200mm, approximately 50mm to 150mm, approximately 50mm to 100mm, approximately 100mm to 500mm, approximately 100mm From approximately 450 mm, from approximately 100 mm to approximately 400 mm, from approximately 100 mm to approximately 350 mm, from approximately 100 mm to approximately 300 mm, from approximately 100 mm to approximately 250 mm, from approximately 100 mm to approximately 200 mm, from approximately 100 mm to approximately 150 mm, from approximately 150 mm to approximately 500 mm, from approximately 150 mm to approximately 450 mm, from approximately 150 mm to approximately 400 mm, from approximately 150 mm to approximately 350 mm, from approximately 150 mm to approximately 300 mm, from approximately 150 mm to approximately 250 mm, from approximately 150 mm to approximately 200 mm, from approximately 200 mm to approximately 500 mm, from approximately 200 mm to approximately 450 mm, from approximately 200 mm to approximately 40 0mM, approximately 200mM to approximately 350mM, approximately 200mM to approximately 300mM, approximately 200mM to approximately 250mM, approximately 250mM to approximately 500mM, approximately 250mM to approximately 450mM, approximately 250mM to approximately 400mM, approximately 250mM to approximately 350mM, approximately 250mM to approximately 300mM, approximately 300mM to approximately 500mM, approximately 300mM to approximately 450mM, approximately 300mM to approximately 400mM, approximately 300mM to approximately 350mM, approximately 350mM to approximately 500mM, approximately 350mM to approximately 450mM, approximately 350mM to approximately 400mM, approximately 400mM to approximately 500mM,It contains at concentrations ranging from approximately 400 mM to approximately 450 mM or approximately 450 mM to approximately 500 mM. In a preferred embodiment, the immunogenic composition contains the excipient at a concentration ranging from approximately 10 mM to approximately 250 mM, most preferably approximately 150 mM.

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

[0323] The compositions may also contain small amounts of wetting, bulking, emulsifying, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, lyophilized powders, or cakes. The formulations should be suitable for the mode of administration. Unless any conventional media or active substance is incompatible with the active ingredient, their use in the immunogenic compositions of the present invention is intended.

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

[0325] In another embodiment, the freeze-dried composition contains at least one sugar in an amount ranging from about 1% (w / v) to about 10% (w / v), for example, 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 freeze-dried composition contains at least one excipient in an amount greater than about 5.5% (w / v), for example, greater than about 7.0% (w / v). In a further embodiment, the lyophilized composition contains about 1% (w / v) to about 10% (w / v) of additional excipients, such as about 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0%. In a preferred embodiment, the lyophilized composition contains 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 additional excipients.

[0326] In another embodiment, the lyophilized composition is redissolved in water, water for injection (WFI), an adjuvant suspension, or physiological 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 isolated polysaccharides and carrier molecules as described herein. Suitable carrier molecules may include 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 polymethyl methacrylate polymers, as well as fine particles derived from poly(lactide) and poly(lactide-co-glycolide), known as poly(lactide) and PLG.

[0328] The immunogenic compositions of the present invention may further comprise one or more additional “immunomodulators” that are active agents that disrupt or modify the immune system, thereby resulting in either upmodulation or downmodulation of humoral and / or cell-mediated immunity. In one particular embodiment, upmodulation of the humoral and / or cell-mediated arms of the immune system is preferred. Examples of certain immunomodulators include, for example, adjuvants or cytokines, or ISCOMATRIX (CSL Limited, Parkville, Australia), as described in U.S. Patent No. 5,254,339. 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 present invention include RIBI adjuvant systems (Ribi Inc., Hamilton, Mont.); mineral gels, e.g., aluminum hydroxide gel; water-in-oil emulsions, e.g., Freund's complete and incomplete adjuvants; block copolymers (CytRx, Atlanta Ga.); SAF-M (Chiron, Emeryville, Calif.); AMPHIGEN® adjuvant; saponins; Quil A or other saponin fractions; monophosphoryl lipids A; and abridine lipid-amine adjuvants. Non-limiting examples of oil-in-water emulsions useful as adjuvants in the vaccine of the present invention include MF59 (U.S. Patent No. 6,299,884) (containing 5% squalene, 0.5% polysorbate 80, and 0.5% Span 85 (which may contain varying amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as the Model 110Y Microfluidizer (Microfluidics, Newton, MA)), and SAF (microfluidized into a submicron emulsion). , either vortexed to produce larger particle size emulsions, containing 10% squalene, 0.4% polysorbate 80, 5% Pluronic® block polymer L121 and thr-MDP); modified SEAM62 (5% (v / v) squalene (Sigma), 1% (v / v) SPAN® 85 surfactant (ICI surfactant), 0.7% (v / v) polysorbate 80 surfactant (ICI surfactant), 2.5% (v / v) ethanol, 200 μg / ml Quill It contains A, 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 the immune response include, but are not limited to, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT) as described in U.S. Patent No. 4,912,094. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkylglucosamine phosphate compounds (AGPs), or derivatives or analogs thereof, available from Corixa (Hamilton, MT) and described in U.S. Patent No. 6,113,918. One such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl (tetra-decanoyoxy-tetrade-canoyl)]-2-[(R)-3-tetradecanoyloxytetradecanoyl-amino]-bD-glucopyranoside, which is also known as 529 (formerly known as RC529). This 529 adjuvant is formulated as an aqueous form (AF) or as a stable emulsion (SE).

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

[0332] Other “immunomodulators” that may be included in the vaccine include, for example, interleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, for example, U.S. Patent No. 5,723,127), 13, 14, 15, 16, 17, and 18 (and their mutant forms); interferon-α, β, and γ; granulocyte-macrophage colony-stimulating factor (GM-CSF) (see, for example, U.S. Patent No. 5,078,996 and ATCC accession number 39900); macrophage colony-stimulating factor (M-CSF); granulocyte colony-stimulating factor (G-CSF); or one or more of tumor necrosis factor α and β. Other adjuvants useful in the immunogenic compositions described herein include, but are not limited to, chemokines encompassing MCP-1, MIP-1α, MIP-1β, and RANTES; adhesion molecules, e.g., selectins, e.g., L-selectin, P-selectin, and E-selectin; mucin-like molecules, e.g., CD34, GlyCAM-1, and MadCAM-1; members of the integrin family, e.g., LFA-1, VLA-1, Mac-1, and p150.95; and members of the immunoglobulin superfamily, e.g., PECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3, etc.; Co-stimulatory molecules, e.g., B7-1, B7-2, CD40 and CD40L, etc.; 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 of whether or not to use immunomodulators and / or adjuvants, or the choice of which immunomodulators and / or adjuvants to use, is expected to depend on the subject to whom 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 the pathogen, an adjuvant may not be necessary because the vaccine antigen can effectively induce a memory response. In certain embodiments, the immunogenic composition will comprise one or more adjuvants. In one embodiment, the immunogenic composition comprises Streptococcus 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 certain embodiments, the adjuvant is aluminum phosphate. In another embodiment of the present invention, the immunogenic composition comprises QS-21 as an adjuvant.

[0334] In one embodiment, the immunogenic composition contains an adjuvant in concentrations ranging from approximately 0.1 mg / ml to approximately 1.0 mg / ml, 0.1 mg / ml to approximately 0.9 mg / ml, 0.1 mg / ml to approximately 0.8 mg / ml, 0.1 mg / ml to approximately 0.7 mg / ml, 0.1 mg / ml to approximately 0.6 mg / ml, 0.1 mg / ml to approximately 0.5 mg / ml, 0.1 mg / ml to approximately 0.4 mg / ml, 0.1 mg / ml to approximately 0.3 mg / ml, 0.1 mg / ml to approximately 0.2 mg / ml, 0.25 mg / ml to approximately 0.95 mg / ml, 0.25 mg / ml to approximately 0.85 mg / ml, 0.25 mg / ml to approximately 0.75 mg / ml, 0.25 mg / ml to approximately 0.65 mg / ml, and 0.25 It contains concentrations ranging from mg / ml to approximately 0.55 mg / ml, 0.25 mg / ml to approximately 0.45 mg / ml, 0.25 mg / ml to approximately 0.35 mg / ml, 0.5 mg / ml to approximately 1.0 mg / ml, 0.5 mg / ml to approximately 0.9 mg / ml, 0.5 mg / ml to approximately 0.8 mg / ml, 0.5 mg / ml to approximately 0.75 mg / ml, 0.5 mg / ml to approximately 0.7 mg / ml, 0.5 mg / ml to approximately 0.65 mg / ml, 0.5 mg / ml to approximately 0.6 mg / ml, 0.75 mg / ml to approximately 1.0 mg / ml, 0.75 mg / ml to approximately 0.95 mg / ml, 0.75 mg / ml to approximately 0.9 mg / ml, and 0.75 mg / ml to approximately 0.85 mg / ml. In a preferred embodiment, the immunogenic composition contains an adjuvant at a concentration ranging from 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, a buffer, a surfactant, an excipient, and optionally an adjuvant, as described herein, 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, the composition being buffered to a pH of about 6.0 to about 7.0, and the capsular polysaccharide having a sialic acid level 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, the composition buffered to a pH of about 6.0 to about 7.0, and the capsular polysaccharide has a sialic acid level of more 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 of histidine, about 0.01% to about 0.03% (v / w) of polysorbate-80, about 10 mM to about 250 mM of sodium chloride, and optionally about 0.25 mg / ml to about 0.75 mg / ml of aluminum as aluminum phosphate, where the capsular polysaccharide has a sialic acid level of more 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, the composition being buffered to a pH of about 6.0 to about 7.0, and the conjugates comprising capsular polysaccharides derived 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 at least two GBS polysaccharide-protein conjugates in concentrations of about 5 mcg / ml to about 50 mcg / ml each, 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 conjugate comprises a capsular polysaccharide derived from at least one additional serotype selected from the group consisting of Group B Streptococcus (GBS) serotype VI and serotypes Ia, Ib, II, III, IV, V, VII, VIII and IX.

[0343] Evaluation of immunogenic compositions The immunogenicity of the immunogenic compositions of the present invention is assessed using various in vitro tests. For example, an in vitro opsonization assay is performed by incubating a mixture of streptococcal cells, thermo-inactivated serum containing specific antibodies against the antigen of concern, and an exogenous complement source. Opsonization phagocytosis proceeds during incubation of newly isolated polymorphonuclear cells (PMNs) or differentiated effector cells such as HL60 and the antibody / complement / streptococcal cell mixture. Bacterial cells coated with antibodies and complement are killed during opsonization phagocytosis. Colony-forming units (cfus) of viable bacteria recovered from opsonization phagocytosis are determined by plate culture of the assay mixture. Titer is reported as the reciprocal of the highest dilution giving 50% bacterial death when determined by comparison with an assay control.

[0344] The in vitro immunogenicity and surface exposure of the antigen may be assessed using a whole-cell ELISA assay. The target bacterial strain (S. agalactiae) is coated onto a plate such as a 96-well plate, and the bacterial cells are reacted with test serum derived from an immunized animal. If antibodies specific to the test antigen are reactive with the antigen's surface exposure epitope, they can be detected by standard methods known to those skilled in the art. Alternatively, flow cytometry may be used to measure the surface exposure of the capsular polysaccharide antigen and the specificity of antibodies, including monoclonal antibodies.

[0345] Next, antigens demonstrating the desired in vitro activity may be tested in an in vivo animal inoculation model. In certain embodiments, the immunogenic composition is used in the immunization of animals (e.g., mice) by immunization methods and routes known to those skilled in the art (e.g., intranasal, parenteral, oral, transrectal, vaginal, percutaneous, intraperitoneal, intravenous, subcutaneous, etc.). After immunization of animals with the GBS immunogenic composition, the animals are inoculated with a strain of Streptococcus agalactiae 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 a desired antigen in an immunogenic composition. Subsequently, the mice are inoculated with S. agalactiae, and their survival is monitored over time after inoculation.

[0347] How to use When used herein, "immunodeficient" refers to a subject suffering from a deficiency with respect to the cellular and / or humoral arms of the immune system. Thus, the degree of deficiency in immune function is intended to range from mild dysfunction in the immune process to complete immunosuppression.

[0348] The term “subject” refers to mammals, birds, fish, reptiles, or any other animal. The term “subject” also includes humans. The term “subject” also includes domestic pets. Non-exclusive examples of domestic 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 domesticated animals. Non-exclusive examples of domesticated animals include alpacas, bison, camels, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

[0349] As used herein, “treatment” (including its variations, e.g., “to treat” or “to be treated”) means one or more of the following: (i) prevention of infection or reinfection, such as with traditional vaccines; (ii) reduction or elimination of the severity of symptoms; and (iii) substantial or complete elimination of the pathogen or disorder in question. Thus, treatment can be achieved prophylactically (before infection) or therapeutically (after infection). In the present invention, prophylactic or therapeutic treatment can be used. According to certain embodiments of the present invention, compositions and methods are provided for treating and immunizing a host animal prophylactically and / or therapeutically against a microbial infection (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 carried out in subjects for biomedical research purposes.

[0350] In another embodiment, the present invention relates to a method for inducing an immune response to GBS in a subject by administering to the subject an effective amount of the immunogenic composition described herein. In one embodiment, the present invention relates to a method for 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 one embodiment, the present invention relates to the immunogenic composition described herein for use as a pharmaceutical. In one embodiment, the present invention relates to the immunogenic composition described herein for use in a method for inducing an immune response to GBS in a subject. In certain embodiments, the subject is a woman planning to become pregnant 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. 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 embodiment, 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 dose of an immunogenic composition can be determined by conducting a dose-response study, in which subjects are immunized with gradually increasing doses of the immunogenic composition, and the immune response is analyzed to determine the optimal dosage. The starting point for this study can be inferred from immunization data in animal models. The dosage may vary depending on the specific conditions of the individual. The dosage can be determined by routine testing using methods known to those skilled in the art.

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

[0353] In one embodiment, patients administered the immunogenic composition of the present invention show a reduction in S. agalactiae carriage rates. Such reduction in carriage or extension of the time interval spent as a non-carrier after administration of the immunogenic composition is significant from the standpoint of medical needs. For example, the overall reduction in S. agalactiae carriage in carriers can be assessed after a single dose of the immunogenic composition of the present 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 the carriage status. The group can then be administered the immunogenic composition of the present invention, with some groups receiving 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, compared to a placebo. One primary endpoint is to compare the carriage rate in patients after administration of the immunogenic composition with placebo at a 3-month interval after immunization.

[0354] antibody An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen-recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless otherwise indicated by the context, the term is intended to encompass not only intact polyclonal or monoclonal antibodies, but also engineered antibodies (e.g., chimeric, humanized, and / or derivatized to alter effector function, stability, and other biological activity) and their fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (ScFv), and domain antibodies (including shark and camel antibodies), as well as fusion proteins containing antibody moieties, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, insofar as they exhibit the desired biological activity), and antibody fragments as described herein, as well as any other modified configurations of immunoglobulin molecules containing antigen-recognition sites. Antibodies can encompass antibodies of any class, e.g., IgG, IgA, or IgM (or their subclasses), and an antibody does not need to belong to any particular class. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. 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 different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known.

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

[0356] The “functional activity” or “functional antibody” of an antibody, as used herein, refers to an antibody that can specifically bind to at least 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 those via opsonization, ADCC, or complement-mediated cytotoxicity. Any subsequent antibody function after antigen binding may be mediated through the Fc region of the antibody. Antibody opsonization phagocytosis assays (OPAs) are in vitro assays designed to measure in vitro Ig complement-assisted killing of bacteria by effector cells (leukocytes), and therefore mimic a biological process. Antibody binding may also directly inhibit the biological function of the antigen to which it is bound. In some embodiments, “functional antibody” refers to an antibody that is functional when measured by an opsonization phagocytosis killing assay that demonstrates bacterial killing in animal efficacy models or that the antibody kills bacteria.

[0357] In one embodiment, the present invention relates to isolated antibodies or fragments thereof that specifically bind to polysaccharides described herein. “Isolated” antibody, as used herein, refers to an antibody identified, separated, and / or recovered from components of its natural environment. Contaminating components of its natural environment are materials that may contain enzymes, hormones, and other proteinaceous or non-proteinaceous solutes that may interfere with the diagnostic or therapeutic use of the antibody. In exemplary embodiments, the antibody will be purified to (1) 95% by weight, most preferably more than 99% by weight, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Since isolated antibodies are expected to be in situ within recombinant cells, at least one component of the antibody’s natural environment is not expected to be present. However, typically, isolated antibodies will be prepared by at least one purification step.

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

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

[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 antibody is 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 antigens of the immunogenic composition of the present invention. In some embodiments, the antibody of the present invention is functional when measured by killing bacteria in any animal efficacy model or via an opsonin phagocytic killing assay. In some embodiments, the antibody of the present invention confers passive immunity to a subject. The present invention further provides polynucleotide molecules encoding the antibody or antibody fragment of the present invention, as well as cells or cell lines (such as hybridoma cells or other engineered cell lines for recombinant antibody production) and transgenic animals that generate the antibody or antibody composition of the present invention using techniques well known to those skilled in the art.

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

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

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

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

[0365] Monoclonal antibodies Antiglycolytic monoclonal antibodies can be prepared by the use of known hybridoma techniques. Typically, the preparation of monoclonal antibodies involves first immunizing a suitable target animal host with a selected immunogen comprising a polysaccharide, oligosaccharide, polysaccharide, or the polysaccharide-protein conjugate of the present invention. Adjuvants, buffers, and / or diluents may be included, if desired. Immunization is carried out in a manner sufficient to guide B lymphocytes to produce or express antibodies that specifically bind to the polysaccharide or its conjugate. Alternatively, lymphocytes are immunized in vitro.

[0366] Next, the lymphocytes are fused with immortalized cell lines using a suitable fusion 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 ("PBLs") are used when human-derived antibodies and cells are desired, while spleen cells or lymph node cells are used when non-human mammalian origin is desired.

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

[0368] Immortalized cell lines are selected based on practical considerations such as origin, fusion, and growth characteristics. For example, suitable immortalized cell lines efficiently fuse, support stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Examples of immortalized cell lines include mouse myeloma cells. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

[0369] Monoclonal antibodies are secreted into the culture medium by 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 hybridoma cells is determined by one of numerous procedures well known to those skilled in the art. For example, antibody binding specificity can be determined by immunoprecipitation, radioimmunoassay (RIA), Western blotting, enzyme-linked immunosorbent assay (ELISA), or surface plasmon resonance (e.g., ViaCore). 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 identifying hybridoma cell-generating antibodies with the desired specificity, the clones are subcloned by limiting dilution and cultured using standard methods. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle medium and RPMI-1640 medium. Alternatively, hybridoma cells are grown in vivo, similar to ascites fluid in mammals. Monoclonal antibodies secreted by the subclones are isolated and purified from the culture medium or ascites fluid using conventional immunoglobulin purification procedures, such as protein A Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

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

[0372] Use of antibodies In one embodiment, the present invention relates to the use of immunogenic compositions described herein for generating GBS antibodies and / or antibody fragments. The polysaccharide-protein conjugates and / or antibodies produced thereby described herein may be used in a variety of immunodiagnostic techniques known to those skilled in the art, including ELISA and microarray-related techniques. In addition, these reagents may be used, for example, to evaluate antibody responses, including serum antibody levels against immunogenic polysaccharide conjugates. The assay methodology of the present invention may involve the use of labeling, such as fluorescence, chemiluminescence, radioactivity, enzyme labeling or dye molecules, and / or secondary immunological reagents for the direct or indirect detection of complexes between antigens or antibodies in a biological sample and corresponding antibodies or antigens bound to a solid support.

[0373] The generated antibodies or antibody fragments may be useful in passive immunotherapy or for the prevention of streptococcal infections.

[0374] Methods for producing polysaccharides In another embodiment, the present invention relates to a method for producing the polysaccharides described herein. The method comprises the steps of culturing GBS and collecting the polysaccharides produced by the bacteria. In one embodiment, GBS comprises S. agalactiae. The bacteria may be any strain of S. agalactiae. In a preferred embodiment, the bacteria are an encapsulated strain of S. agalactiae. The S. agalactiae strains used in this invention are 090, A909 (ATCC accession number BAA-1138), 515 (ATCC accession number BAA-1177), B523, CJB524, MB4052 (ATCC accession number 31574), H36B (ATCC accession number 12401), S40, S42, MB4053 (ATCC accession number 31575), M709, 133, 7 357, PFEGBST0267, MB4055 (ATCC accession number 31576), 18RS21 (ATCC accession number BAA-1175), S16, S20, V8 (ATCC accession number 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB4082 (ATCC accession number 31577), M132, 110, M781 (ATCC accession number BAA-22), D136C(3)(AT CC accession number 12403), M782, S23, 120, MB4316 (M-732; ATCC accession number 31475), M132, K79, COH1 (ATCC accession number BAA-1176), PFEGBST0563, 3139 (ATCC accession number 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC accession number BAA-23), CJB112, 2603 This includes V / R (ATCC accession number 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 may include Columbian broth. The medium may also include dextrose, hemin, and / or glucose. In one embodiment, the medium includes Columbian broth and dextrose. When S. agalactiae is cultured using Columbian broth and dextrose, the temperature for cultivation is preferably 20 to 40°C, preferably 37°C. In a preferred embodiment, the bacteria are cultured under aerobic conditions. In another preferred embodiment, the bacteria are cultured for 12 to 60 hours.

[0376] Polysaccharides can be collected from the obtained culture by using methods known to those skilled in the art for collecting target substances from the culture, such as heating, enzymatic treatment, centrifugation, precipitation, treatment with activated carbon, and / or filtration (see, for example, U.S. Patent Applications Publications 2006 / 0228380, 2006 / 0228381, 2007 / 0184071, 2007 / 0184072, 2007 / 0231340, and 2008 / 0102498; International Patent Application Publication WO2008 / 118752). In one embodiment, the culture containing bacteria and polysaccharides is centrifuged and treated with enzymes, such as lysozyme, RNase, DNase, pronase, mutanolicin, 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. Next, the polysaccharides may be precipitated by adding a suitable organic solvent to the supernatant, and the polysaccharides may be collected by centrifugation. More specifically, the polysaccharides described herein can be obtained by adding ethanol to the supernatant from which bacteria have been removed at a final concentration of about 25% by volume, removing the protein-containing precipitate by centrifugation, adding ethanol to a final concentration of about 75% by volume, and then collecting the precipitate by centrifugation. The obtained precipitate may be dried under nitrogen. The obtained precipitate may be resuspended in Tris and 0.05% sodium azide.

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

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

[0379] In one embodiment of the present invention, the hydroxylamine may be one of those listed in Table 2 of Example 2. In a preferred embodiment, the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine, diethylhydroxylamine, hydroxylamine, ethylenediamine, triethylenetetramine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, and 2,6,10,trimethyl2,6,10-triazoundecane.

[0380] In one embodiment of the present invention, the concentration of hydroxylamine is about 5 mM to about 200 mM, for example, about 5 mM to about 150 mM, about 5 mM to about 100 mM, about 5 mM to about 75 mM, about 5 mM to about 50 mM, about 5 mM to about 25 mM, about 5 mM to about 10 mM, 10 mM to about 200 mM, for example, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 1 These ranges include 0 mmM to approximately 75 mmM, approximately 10 mmM to approximately 50 mmM, approximately 10 mmM to approximately 25 mmM, approximately 25 mmM to approximately 200 mmM, approximately 25 mmM to approximately 150 mmM, approximately 25 mmM to approximately 100 mmM, approximately 25 mmM to approximately 75 mmM, approximately 25 mmM to approximately 50 mmM, approximately 50 mmM to approximately 200 mmM, approximately 50 mmM to approximately 150 mmM, 50 mmM to approximately 100 mmM, and approximately 50 mmM to approximately 75 mmM, etc.

[0381] In another aspect, the pH of the reaction is maintained between approximately 5.5 and approximately 9.5, for example, approximately 5.5 to approximately 9.0, approximately 5.5 to approximately 8.5, approximately 5.5 to approximately 8.0, approximately 5.5 to approximately 7.5, approximately 5.5 to approximately 7.0, approximately 5.5 to approximately 6.5, approximately 6.0 to approximately 9.5, approximately 6.0 to approximately 9.0, approximately 6.0 to approximately 8.5, approximately 6.0 to approximately 8.0, approximately 6.0 to approximately 7.5, approximately 6.0 to approximately 7.0, approximately 6.5 to approximately 9.5, approximately 6.5 to approximately 8.5, approximately 6.5 to approximately 8.0, approximately 6.5 to approximately 7.5, approximately 7.0 to approximately 9.5, approximately 7.0 to approximately 9.0, 7.0 to approximately 8.5, and approximately 7.0 to approximately 8.0, etc.

[0382] A further aspect of the present invention is that the reaction takes place at temperatures ranging from about 20°C to about 85°C, for example, about 20°C to about 80°C, about 20°C to about 75°C, about 20°C to about 70°C, about 20°C to about 65°C, about 20°C to about 60°C, about 20°C to about 55°C, about 20°C to about 50°C, about 25°C to about 85°C, about 25°C to about 80°C, about 25°C to about 75°C, and about 25°C. From approximately 70°C, from approximately 25°C to approximately 65°C, from approximately 25°C to approximately 60°C, from approximately 25°C to approximately 55°C, from approximately 25°C to approximately 50°C, from approximately 30°C to approximately 85°C, from approximately 30°C to approximately 80°C, from approximately 30°C to approximately 75°C, from approximately 30°C to approximately 70°C, from approximately 30°C to approximately 65°C, from approximately 30°C to approximately 60°C, from approximately 30°C to approximately 55°C, from approximately 30°C to approximately 50°C, and from approximately 35°C to approximately 85°C. Approximately 35°C to 80°C, approximately 35°C to 75°C, approximately 35°C to 70°C, approximately 35°C to 65°C, approximately 35°C to 60°C, approximately 35°C to 55°C, approximately 40°C to 85°C, approximately 40°C to 80°C, approximately 40°C to 75°C, approximately 40°C to 70°C, approximately 40°C to 65°C, approximately 40°C to 60°C, approximately 45°C to 85°C, approximately 45°C It occurs at temperatures such as from approximately 80°C, from approximately 45°C to approximately 75°C, from approximately 45°C to approximately 70°C, from approximately 45°C to approximately 65°C, from approximately 50°C to approximately 85°C, from approximately 50°C to approximately 80°C, from approximately 50°C to approximately 75°C, from approximately 50°C to approximately 70°C, from approximately 55°C to approximately 85°C, from approximately 55°C to approximately 80°C, from approximately 55°C to approximately 75°C, from approximately 60°C to approximately 85°C, and from approximately 65°C to approximately 85°C.

[0383] Another type of reaction time is approximately 10 to 90 hours, for example, approximately 10 to 85 hours, approximately 10 to 80 hours, approximately 10 to 75 hours, approximately 10 to 70 hours, approximately 10 to 60 hours, approximately 10 to 50 hours, approximately 10 to 40 hours, approximately 10 to 30 hours, approximately 10 to 25 hours, approximately 10 to 20 hours, approximately 10 to 15 hours, approximately 15 to 90 hours, approximately 15 to 85 hours, approximately 15 to 80 hours, and approximately 15 hours to These range from approximately 75 hours, 15 to 70 hours, 15 to 60 hours, 15 to 50 hours, 15 to 40 hours, 15 to 30 hours, 15 to 20 hours, for example, 20 to 90 hours, 20 to 85 hours, 20 to 80 hours, 20 to 75 hours, 20 to 70 hours, 20 to 60 hours, 20 to 50 hours, 20 to 40 hours, 20 to 30 hours, and 20 to 25 hours, etc.

[0384] Alternatively, in another embodiment of the present invention, the polysaccharide is chemically synthesized. The polysaccharide can be chemically synthesized according to conventional methods.

[0385] In yet another embodiment of the present 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 polysaccharide described herein, and the repeating units of the polysaccharide produced in the host cell are partially identical to the repeating units of the polysaccharide described herein. The polysaccharide is structurally similar to the polysaccharide described herein, and for example, if the repeating units of the polysaccharide have unknown branching, they are heterogeneous in size and / or heterogeneous in branching arrangement compared to the repeating units of the polysaccharide described herein. Preferably, the host cell is a bacterial host cell. [Examples]

[0386] The following examples illustrate some embodiments of the present invention. However, it should be understood that these examples are for illustrative purposes only and do not claim to be entirely definitive regarding the conditions and scope of the present invention. It should be understood that, while generally less convenient, conditions both above and below the specified range can also be used when typical reaction conditions (e.g., temperature, reaction time, etc.) are given. All parts and percentages mentioned herein are on a weight basis, and all temperatures are expressed in degrees Celsius unless otherwise specified.

[0387] Furthermore, unless otherwise described in detail, the following examples were performed using standard techniques that are well known and routine to those skilled in the art. As noted above, the following examples are provided for illustrative purposes only 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 of each serotype were fermented in pH-controlled deep culture in a limited medium. The procedures and media used were optimized through experimentation and were extensions of the basic techniques previously described by von Hunolstein, C. et al., Appl. Micro. Biotech. 38(4):458-462 (1993). Capsular polysaccharides were removed from the cells by NaOH treatment. After purification, purified polysaccharides were obtained by a series of UF / DF, precipitation, and carbon filtration steps. See, for example, U.S. Patent No. 8,652,480. Activated polysaccharides were converted to CRM using reductive amination chemistry. 197 They were conjugated together. See, for example, U.S. Patent No. 5,360,897.

[0389] (Example 2) Conjugation of GBS capsule polysaccharides by reductive amination Activating polysaccharides Polysaccharide oxidation was carried out by sequentially adding calculated amounts of 500 mM potassium phosphate buffer (pH 6.0) and sterile water for injection (WFI) to 100 mM potassium phosphate buffer (pH 6.0 ± 0.5) 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. Oxidation was initiated by adding 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 activated polysaccharides were performed using a 5K MWCO ultrafiltration cassette. Hemodiafiltration was performed against a 20x diavolume WFI. The purified activated polysaccharides were then stored at 5±3°C. The purified activated sugars were characterized, in particular, by (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 of activated polysaccharides (DO = moles of sugar repeating units / moles of aldehydes) 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. In the anthrone method, polysaccharides are first broken down into monosaccharides by the action of sulfuric acid and heat. The anthrone reagent reacts with hexose to form a yellow-green complex, the absorbance of which is read spectrophotometrically at 625 nm. Within the assay range, the absorbance is directly proportional to the amount of hexose present.

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

[0394] The activated polysaccharide is combined with a sucrose excipient and freeze-dried. The activated polysaccharide was mixed with sucrose at a ratio of 25 grams of sucrose per gram of activated polysaccharide. The bottles containing the mixture were then freeze-dried. After freeze-drying, the bottles containing the freeze-dried activated polysaccharide were stored at -20±5°C. (Calculated CRM) 197 The protein was shell-frozen and freeze-dried separately. Freeze-dried CRM 197 It was stored at -20±5℃.

[0395] Redissolve freeze-dried activated polysaccharides and carrier proteins. The freeze-dried 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 freeze-dried CRM. 197 It was added to redissolve it.

[0396] Conjugate and cap Redissolved activated polysaccharides, redissolved CRM 197The two components were combined in a reaction vessel, followed by thorough mixing to obtain a clear solution, after which conjugation with sodium cyanoborohydride was initiated. The final polysaccharide concentration in the reaction solution was approximately 1 g / L. 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 hours. The conjugation reaction was terminated by adding 2 MEq of sodium borohydride (NaBH4) to cap the unreacted aldehyde. This capping reaction was continued at 23±2°C for 3±1 hours.

[0397] Purify the conjugate For purification of the conjugate solution by tangent flow filtration using a 100-300K MWCO membrane, the prepared product was diluted 1:10 with chilled 5 mM succinate-0.9% physiological saline (pH 6.0).

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

[0399] (Example 3) Conjugation of GBS capsule polysaccharides by CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) GBS polysaccharide was dialyzed against WFI to remove buffer salts and then lyophilized. The 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 physiological saline). The pH of the reaction mixture was adjusted to 8.8 and stirred at room temperature for 24 hours. The conjugation reaction mixture was quenched with 2 M glycine, the conjugate was passed through a 5 μm filter, and purified with physiological saline by tangent flow filtration using a 100 K MWCO membrane. The final 0.22 μm filtration step was completed to obtain the immunogenic conjugate.

[0400] (Example 4) GBS polysaccharide-CRM 197 The effect of various conjugation conditions on conjugates GBS serotypes VI, VII, VIII, and IX conjugates can be produced by deliberately varying periodic acid oxidation / reductive amination (PO / RAC) conditions, including the solvent for the reagent (aqueous medium vs. DMSO), varying levels of sialic acid in the initial polysaccharide, and degree of oxidation / sugar epitope modification. Generally, 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 medium.

[0401] Conjugation processes that produce conjugates with low levels of unreacted (free) polysaccharides are advantageous and preferred. It is well known that high levels of unreacted (free) polysaccharides can lead to an excessive T cell-independent immune response, which may 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 desiallated using methods known in the art (see Chaffin, DO et al., J Bacteriol 187(13):4615~4626 (2005)) to produce conjugate mutants, and the effect of desiallation percentage on immunogenicity can be determined. Generally, desiallation of more than about 40% (i.e., sialic acid level less than about 60%) adversely affects immunogenicity.

[0403] Similarly, in most cases, oxidation degrees below approximately 5, or sugar epitope modifications above approximately 20%, have adverse effects on immunogenicity. Since oxidation occurs via sialic acid on the capsular polysaccharide, it can be seen that sugar epitope modifications above approximately 20% reduce the sialic acid content, which leads to reduced immunogenicity.

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

[0405] Additional conjugate variants can also be produced using alternative chemical pathways. One alternative chemistry involves producing a conjugate by reacting a polysaccharide with carbonyl ditriazole (CDT) and carrying out the conjugation reaction in DMSO. In another alternative chemistry, the conjugate can be produced as detailed in the above examples by oxidation of the polysaccharide using the reagent TEMPO[(2,2,6,6-tetramethylpiperidine-1-yl)oxyl] (instead of sodium periodate), followed by conjugation using reductive amination chemistry (TEMPO / RAC) in DMSO. Conjugates produced by these alternative chemistry methods have been shown to be immunogenic in mice and to be compatible with alternative chemical pathways other than PO / RAC. However, some conjugation chemistry can be performed better with certain serotypes than others.

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

[0407] (Example 5) The monovalent conjugate vaccine generated functional and conjugated antibody responses in rabbits. Rabbits (4-5 rabbits per group) were vaccinated at weeks 0, 3, and 6 with 20 mcg of monovalent conjugate polysaccharides of GBS CPS serotype VI, VII, VIII, or IX, based on the weight of the polysaccharide conjugate, which was formulated with 20 mcg of QS21 per conjugate dose and conjugated with different carrier proteins (CRM197, tetanus toxoid (TT), SCP). Serum was assessed for anti-CPS IgG titer at baseline and at week 10 by direct-binding Luminex immunoassay (dLIA) using CPS-coated microspheres.

[0408] Figure 1 shows the mean binding activity profiles of serially diluted serum from two rabbits administered with the GBS CPS serotype VI-CRM197 conjugate. The interpolated EC50 serum dilution titers for GBS CPS VI shown in Figure 1 were determined using sigmoid dose-response curve fitting (slope with changing gradient) (graph pad 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 to serotype VI, and when assessed by direct-binding Luminex immunoassay, serotype VI-induced serum showed only minimal binding to other serotypes.

[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 induced robust IgG antibody titers against immunized congener polysaccharides.

[0411] [Table 2]

[0412] Figures 3A and 3B show the mean binding activity profiles of serially diluted serum from two rabbits administered with 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 sigmoid dose-response curve fitting (slope with changing gradient) (graph pad prism).

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

[0414] [Table 3]

[0415] Rabbit-derived serum immunized with GBS VI-CRM197 or VI:SCP demonstrated OPA-mediated kill activity against target strains expressing serotype VI capsules. Pre-adsorption of GBS VI-SCP-induced serum with soluble SCP protein prior to OPA analysis slightly reduced the observed titer, demonstrating specificity. Very slight cross-reactivity killing with GBS VI-CRM197-induced serum was observed against target strains expressing heterologous serotypes III, VIII, or IX. In comparison, serum from rabbits immunized with serotype GBS VI-SCP demonstrated measurable titer against strains expressing heterologous serotypes III, VIII, or IX. Adsorption of SCP-specific antibodies with soluble SCP effectively reduced the observed titer, supporting the ability of anti-SCP antibodies to achieve functional activity across isolates in the context of diverse capsule types.

[0416] These results indicate that the functional activity of anti-CPS antibodies is primarily limited to allospecific serotypes, and that anti-SCP carrier antibodies can confer moderate but broad levels of CPS-independent cross-functional activity across diverse isolates expressing different CPS types.

[0417] (Example 6) The multivalent conjugate vaccine induced a binding antibody response in rabbits. Single rabbits were vaccinated at weeks 0, 3, 6, and 9 with CRM197 conjugates of serotypes Ia, Ib, II, III, IV, V, and VI, using 20 mcg doses of each antigen, and administered 20 mcg of QS21 adjuvant. Serum IgG titers were assessed at week 11 using the Luminex assay described in Example 5. Serum 3-fold serial dilutions were tested starting at 1:15.

[0418] Figure 4 shows the serological responses to the seven administered serotypes Ia, Ib, II, III, IV, V, and VI-CRM197 conjugates (open symbol) and three serotypes (VII, VIII, IX) (closed symbol) that are not present in the formulation. The results demonstrate that the hepatovalent GBS glycoconjugate vaccine, which includes the newly emerging serotype VI, can induce antigen-specific IgG antibodies against all seven capsular polysaccharide components. The responses to serotype VII and IX polysaccharides may be due to cross-reactivity of antibodies against the serotype V antigen, sharing a common epitope within the branched sialyzed side chains of the repeating units (Berti, F. et al., (2014) JBC 289:34 23437~23448).

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

[0420] C1. An immunogenic polysaccharide-protein conjugate comprising Group B Streptococcus (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of more than approximately 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, with a capsular polysaccharide of serotype VI.

[0423] C4. An immunogenic conjugate of C2, with a capsular polysaccharide of serotype VII.

[0424] C5. An immunogenic conjugate of C2, with a capsular polysaccharide of serotype VIII.

[0425] C6. An immunogenic conjugate of C2, with a capsular polysaccharide of serotype IX.

[0426] C7. An immunogenic conjugate of any one of the C1-C6 compounds, in which the capsular polysaccharide has a sialic acid level of over 95%.

[0427] C8. An immunogenic conjugate of any one of the C1-C7 compounds, in which the capsular polysaccharide has approximately 100% sialic acid levels.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0441] C22. An immunogenic conjugate of any one of the C1-21 terms, with a conjugate molecular weight between approximately 300 kDa and approximately 20,000 kDa.

[0442] C23. An immunogenic conjugate of any one of the C1-C22 terms, with a conjugate molecular weight between approximately 1,000 kDa and approximately 15,000 kDa.

[0443] C24. An immunogenic conjugate of any one of the C1-C23 terms, with a conjugate molecular weight between approximately 1,000 kDa and approximately 10,000 kDa.

[0444] C25. An immunogenic conjugate of any one of the C1-C24 terms, in which each polysaccharide is independently conjugated with a carrier protein.

[0445] C26. Carrier protein, CRM 197Alternatively, an immunogenic conjugate of any one of the C1-C25 categories, which is a tetanus toxoid.

[0446] C27. Carrier protein, CRM 197 An immunogenic conjugate of any one of the C1-C26 terms.

[0447] C28. A method for isolating a capsular polysaccharide, comprising the step of 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. A method of killing bacteria by heat, similar to C28 or C29.

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

[0451] C32. The method of any one of items C28-C31, further comprising the step of filtering.

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

[0453] C34. The bacterium is Streptococcus agalactiae, as in method C28.

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

[0455] C36. An immunogenic composition comprising a polysaccharide-protein conjugate, wherein the conjugate comprises a capsular polysaccharide derived from Group B Streptococcus (GBS) serotype VI, which is conjugated with a carrier protein.

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

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

[0458] C39. An immunogenic composition of C38 further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype Ib.

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

[0460] C41. An immunogenic composition according to any one of C38 to C40, further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype III.

[0461] C42. An immunogenic composition according to any one of C38 to C41, further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype IV.

[0462] C43. An immunogenic composition according to any one of C38 to C42, further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype V.

[0463] C44. An immunogenic composition according to any one of C38 to C43, further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype VII.

[0464] C45. An immunogenic composition according to any one of C38 to C44, further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype VIII.

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

[0466] C47. An immunogenic composition of C36 further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype VII.

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

[0468] C49. An immunogenic composition according to any one of C46 to C48, further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype IX.

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

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

[0471] C52. An immunogenic composition according to either C50 or C51, further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype IX.

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

[0473] C54. An immunogenic composition of C53 further comprising a conjugate containing a capsular polysaccharide derived from GBS serotype IX.

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

[0475] C56. An immunogenic composition comprising a polysaccharide-protein conjugate containing 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. An immunogenic composition of C56 comprising at least two GBS capsular polysaccharide serotypes.

[0477] C58. An immunogenic composition of C56 containing at least three GBS capsular polysaccharide serotypes.

[0478] C59. An immunogenic composition of C56 containing at least four GBS capsular polysaccharide serotypes.

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

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

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

[0482] C63. An immunogenic composition of C56 containing at least eight GBS capsular polysaccharide serotypes.

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

[0484] C65. An immunogenic composition according to any one of C35 to C64, further comprising pharmaceutically acceptable excipients, buffers, stabilizers, adjuvants, antifreeze agents, salts, divalent cations, nonionic surfactants, free radical oxidation inhibitors, carriers, or mixtures thereof.

[0485] C66. An immunogenic composition according to any one of C35 to C65, further comprising a buffering agent.

[0486] C67. An 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. An immunogenic composition of C67, wherein the buffering agent is histidine.

[0488] C69. An immunogenic composition according to any one of C35 to C68, further comprising a surfactant.

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

[0490] C71. An immunogenic composition of C70 in which the surfactant is polysorbate-80.

[0491] C72. An immunogenic composition according to any one of C35 to C71, further comprising an excipient.

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

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

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

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

[0496] C77. An 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. An immunogenic composition of C77, wherein the adjuvant is aluminum phosphate.

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

[0499] C80. An immunogenic composition according to any one of items C35 to 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. An immunogenic composition according to any one of items C35 to C80, comprising histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, and buffered to a pH of about 6.0 to about 7.0.

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

[0502] C83. An immunogenic composition according to any one of items C35 to C82, containing doses ranging from approximately 5 mcg / ml to approximately 50 mcg / ml.

[0503] C84. An immunogenic composition according to any one of C35 to C83, which is lyophilized in the presence of at least one excipient, if applicable.

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

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

[0506] C87. An immunogenic composition according to any one of C84 to C86, comprising at least one excipient in an amount of approximately 1% (w / v) to approximately 10% (w / v).

[0507] C88. An immunogenic composition according to any one of items C84 to C87, comprising additional excipients.

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

[0509] C90. An immunogenic composition of C88 or C89 containing approximately 1% (w / v) to approximately 10% (w / v) of additional excipients.

[0510] C91. An immunogenic composition according to any one of items C84 to C90, which can be redissolved in water, water for injection (WFI), adjuvant suspension, or physiological saline.

[0511] C92. An immunogenic composition according to any one of items C44 to C91, for use as a pharmaceutical.

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

[0513] C94. An immunogenic composition of C93, for use in women planning pregnancy or pregnant women.

[0514] C95. An immunogenic composition of C94 in a woman in the latter half of pregnancy.

[0515] C96. An immunogenic composition of C95 in a pregnant woman who is at least 20 weeks pregnant.

[0516] C97. An immunogenic composition of C96 for pregnant women who are 27 to 36 weeks pregnant.

[0517] C98. An immunogenic composition of C97, for use in adults aged 50 years or older.

[0518] C99. An immunogenic composition of C98, for use in adults aged 65 years or older.

[0519] C100. An immunogenic composition of C99, for use in adults aged 85 years or older.

[0520] C101. An immunogenic composition according to any one of the items C90 to C100, for which the target is immunodeficient.

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

[0522] C103. An immunogenic composition according to any one of items C93 to C102, wherein the Group B Streptococcus is Streptococcus agalactiae.

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

[0524] C105. A method for 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 according to any one of C35 to C104.

[0525] C106. The method of C104 or C105, wherein the subjects are women who are planning to become pregnant or are pregnant.

[0526] C107. The method used in C106 for women in the latter half of pregnancy.

[0527] C108. A pregnant woman, at least 20 weeks pregnant, using the method described in C106 or C107.

[0528] C109. A pregnant woman who is between 27 and 36 weeks pregnant, using any one of the methods described in C106-C108.

[0529] C110. The method of C104 or C105, wherein the subjects are adults aged 50 or older.

[0530] C111. The same method as C110, but with the subjects being adults aged 65 or older.

[0531] C112. The method of C110 or C111, wherein the subjects are adults aged 85 years or older.

[0532] C113. A method described in any one of the items C104-C112, wherein the subject is immunocompromised.

[0533] C114. The method of C113, wherein the subjects have 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 a capsular polysaccharide in any one of the immunogenic conjugates described in C1 to C27.

[0536] A composition containing antibodies to C117 and C116.

[0537] C118. A method for conferring passive immunity to a target, (a) A step of producing an antibody preparation using an immunogenic composition of any preceding paragraph, (b) A step of administering an antibody preparation to the target to confer passive immunity. A method that includes this.

[0538] C119. A method for producing an immunogenic polysaccharide-protein conjugate according to any one of the items C1 to C27, (a) A step of reacting GBS capsule polysaccharide with an oxidizing agent to obtain an activated polysaccharide, (b) A step in which the activated polysaccharide is reacted with a carrier protein to yield a polysaccharide-protein conjugate. A method that includes this.

[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 dimethyl sulfoxide (DMSO), sulfolane, dimethylformamide (DMF), and hexamethylphosphoramide (HMPA).

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

[0542] C123. A method according to any one of items C119 to C122, in which a polysaccharide is reacted with 0.01 to 10.0 molar equivalents of an oxidizing agent.

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

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

[0545] C126. The method of any one of C119-C125, wherein the oxidation reaction in step (a) takes between 1 and 50 hours.

[0546] C127. A method according to any one of items C119-C126, wherein the oxidation reaction temperature is maintained between approximately 2°C and approximately 25°C.

[0547] C128. The method according to any one of items C119 to 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 between approximately 1 mM and approximately 500 mM.

[0549] C130. A method according to any one of the C119-C129 items, in which the oxidation reaction is carried out at a pH between approximately 4.0 and approximately 8.0.

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

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

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

[0553] C134. A method according to any one of items C119 to C133, wherein the concentration of the polysaccharide is between approximately 0.1 mg / mL and approximately 10.0 mg / mL.

[0554] C135. A method according to any one of the items in C119-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 freeze-drying an activated polysaccharide.

[0556] C137. The method of C136, wherein the activated polysaccharide is freeze-dried in the presence of a sugar selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melegitose, dextran, mannitol, lactitol, and palatinite.

[0557] C138. Step (b) is, (1) A step of combining activated polysaccharide with a carrier protein, (2) The step of reacting the formulated activated polysaccharide and carrier protein with a reducing agent to form a GBS capsule polysaccharide-carrier protein conjugate. A method according to any one of the terms C119-C137, including the method described in C119-C137.

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

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

[0560] C141. Reducing agents include Brønsted or Lewis acid, pyridineborane, 2-picolinborane, 2,6-diborane-methanol, dimethylamine-borane, and t-BuMe i A method according to any one of items C138 to C139, 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-methylpyridineborane (PEMB).

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

[0562] C143. A method according to any one of items C138 to C142, wherein the amount of reducing agent is between approximately 0.1 and approximately 10.0 molar equivalents.

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

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

[0565] C146. The method according to any one of C119 to C145, further comprising the step of capping unreacted aldehydes by adding boron hydride (step (c)).

[0566] C147. The method of C146, wherein the amount of boron hydride is between approximately 0.1 and approximately 10.0 molar equivalents.

[0567] C148. The method of C146, wherein boron hydride 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, where boron hydride 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 according to any one of C146-C149, wherein the temperature of the capping step is maintained between approximately 15°C and approximately 45°C.

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

[0572] C153. A method according to any one of items C119 to C152, wherein the polysaccharide-protein conjugate contains less than 40% free polysaccharides compared to the total amount of polysaccharides.

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

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

[0575] C156. A method for producing polysaccharide-protein conjugates, (a) A step of reacting isolated GBS capsule polysaccharide with an oxidizing agent, (b) The step of quenching the oxidation reaction product from step (a) by adding a quenching agent to obtain activated GBS capsule polysaccharide, (c) A step of combining activated GBS capsule polysaccharide with a carrier protein, (d) The step of reacting the formulated activated GBS capsule polysaccharide and carrier protein with a reducing agent to form a GBS capsule polysaccharide-carrier protein conjugate, (e) A step in which unreacted aldehydes are capped by the addition of sodium borohydride (NaBH4) Includes, Here, steps (c) and (d) are performed in DMSO.

[0576] References 1. Paoletti, LJ, J. Bradford, and LC Paoletti, "Serotype VIII Strain among Colonizing Group B Streptococcus 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 from 2011 to 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 non-invasive Group B Streptococcus 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 Highly Virulent Isolates Causing Neonatal Invasive Diseases in Southern Mainland China." Streptococcus CC17 Hypervirulent Isolates Causing Neonatal Invasive Disease in Southern Mainland China.)” 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): pp. 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 Diseases in Neonatal and Adults" Streptococcus Strains Causing Invasive Disease in Neonates and Adults.)” Frontiers in microbiology, 2019. 10: p. 264-264.

Claims

[Claim 1] An immunogenic composition comprising a polysaccharide-protein conjugate containing serotype VI group B streptococcus (GBS) capsular polysaccharide 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 III, VIII, or IX.

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