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

Abstract

The invention relates to 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 the 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 also relates to immunogenic compositions comprising polysaccharide-protein conjugates, wherein the conjugates comprise a CP from GBS serotype IV and at least one additional serotype. 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. The resulting antibodies can be used to treat or prevent GBS infection via passive immunotherapy.

1 . An immunogenic polysaccharide-protein conjugate comprising a group B  streptococcus  (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level of greater than about 60%, greater than about 95%, or about 100%. 
     
     
         2 . The immunogenic conjugate of  claim 1 , wherein the capsular polysaccharide is selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX. 
     
     
         3 . The immunogenic conjugate of  claim 1 , wherein the capsular polysaccharides has at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 mM sialic acid per mM of polysaccharide. 
     
     
         4 . The immunogenic conjugate of  claim 1 , wherein the capsular polysaccharide has a molecular weight of between about 5 kDa and about 1,000 kDa; between about 25 kDa and about 750 kDa; between about 25 kDa and about 400 kDa; between about 25 kDa and about 200 kDa; or between about 100 kDa and about 400 kDa. 
     
     
         5 . The immunogenic conjugate of  claim 1 , wherein the molecular weight of the conjugate is between about 300 kDa and about 20,000 kDa; between about 1,000 kDa and about 15,000 kDa; or between about 1,000 kDa and about 10,000 kDa. 
     
     
         6 . The immunogenic conjugate of  claim 1 , wherein the capsular polysaccharide is between about 0% and about 40% O-acetylated. 
     
     
         7 . The immunogenic conjugate  claim 1 , wherein the capsular polysaccharide is less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% O-acetylated. 
     
     
         8 . The immunogenic conjugate of  claim 1 , wherein the capsular polysaccharide has at least about 0.1, 0.2, 0.3, 0.35 or about 0.4 mM O-acetate per mM saccharide repeating unit. 
     
     
         9 . The immunogenic conjugate of  claim 1 , wherein the carrier protein is CRM 197  or tetanus toxoid. 
     
     
         10 . The immunogenic conjugate of  claim 9 , wherein the carrier protein is CRM 197 . 
     
     
         11 . An immunogenic composition comprising the immunogenic polysaccharide-protein conjugate of  claim 1 . 
     
     
         12 . The immunogenic composition of  claim 11 , wherein the composition further comprises a pharmaceutically acceptable excipient, buffer, stabilizer, adjuvant, a cryoprotectant, a salt, a divalent cation, a non-ionic detergent, an inhibitor of free radical oxidation, a carrier, or a mixture thereof. 
     
     
         13 . The immunogenic composition of  claim 12 , wherein the composition comprises a buffer. 
     
     
         14 . The immunogenic composition of  claim 13 , wherein the buffer is selected from the group consisting of HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. 
     
     
         15 . The immunogenic composition of  claim 12 , wherein the composition comprises a surfactant. 
     
     
         16 . The immunogenic composition of  claim 15 , wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20, and polyoxyethylene alkyl ethers. 
     
     
         17 . The immunogenic composition of  claim 12 , wherein the composition comprises an excipient. 
     
     
         18 . The immunogenic composition of  claim 17 , wherein the excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), dried skim milk, glycerol, propylene glycol, water, and ethanol. 
     
     
         19 . The immunogenic composition of  claim 12 , wherein the composition comprises an adjuvant. 
     
     
         20 . The immunogenic composition of  claim 19 , wherein the adjuvant is an aluminum-based adjuvant or QS-21. 
     
     
         21 . The immunogenic composition of  claim 12 , wherein the composition comprises a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the composition is buffered to a pH of about 6.0 to about 7.0. 
     
     
         22 . The immunogenic composition of  claim 11 , wherein the composition comprises a dose of about 5 mcg/ml to about 50 mcg/ml. 
     
     
         23 . The immunogenic composition of  claim 11 , wherein the composition is lyophilized, optionally in the presence of at least one excipient. 
     
     
         24 . The immunogenic composition of  claim 23 , wherein the at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), dried skim milk, glycerol, propylene glycol, water, and ethanol. 
     
     
         25 . The immunogenic composition of  claim 24 , wherein the at least one excipient is sucrose. 
     
     
         26 . The immunogenic composition of  claim 23 , wherein the composition comprises an additional excipient. 
     
     
         27 . The immunogenic composition of  claim 26 , wherein the additional excipient is mannitol or glycine. 
     
     
         28 . A method of inducing an immune response against group  B. streptococcus  comprising administering to a subject an effective amount of the immunogenic composition of  claim 11 . 
     
     
         29 . A method of preventing or reducing a disease or condition associated with group  B. streptococcus  in a subject comprising administering to a subject an effective amount of the immunogenic composition of  claim 11 . 
     
     
         30 . A method of producing an antibody comprising administering the immunogenic composition of  claim 11  to a subject. 
     
     
         31 . An antibody produced by the method of  claim 30 . 
     
     
         32 . A method of conferring passive immunity to a subject comprising the steps of:
 (a) generating an antibody preparation using the immunogenic composition of  claim 11 ; and   (b) administering the antibody preparation to the subject to confer passive immunity.   
     
     
         33 . An antibody that binds to a capsular polysaccharide in the immunogenic conjugate of  claim 1 . 
     
     
         34 . A composition comprising the antibody of  claim 33 . 
     
     
         35 . A method of making an immunogenic polysaccharide-protein conjugate of  claim 1  comprising the steps of:
 (a) reacting the GBS capsular polysaccharide with an oxidizing agent resulting in an activated polysaccharide; and 
 (b) reacting the activated polysaccharide with the carrier protein resulting in a polysaccharide-protein conjugate. 
 
     
     
         36 . A method of making a polysaccharide-protein conjugate comprising the steps of:
 (a) reacting isolated GBS capsular polysaccharide with an oxidizing agent;   (b) quenching the oxidation reaction of step (a) by addition of a quenching agent resulting in an activated GBS capsular polysaccharide;   (c) compounding the activated GBS capsular polysaccharide with a carrier protein,   (d) reacting the compounded activated GBS capsular polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate, and   (e) capping unreacted aldehyde by addition of sodium borohydride (NaBH 4 ), wherein steps (c) and (d) are carried out in DMSO.   
     
     
         37 . A method of isolating a capsular polysaccharide comprising reacting an organic reagent with a cell broth comprising a capsular polysaccharide producing bacterium. 
     
     
         38 . A method of making an immunogenic polysaccharide-protein conjugate, wherein the capsular polysaccharide is isolated according to the method of  claim 37 . 
     
     
         39 . An immunogenic polysaccharide-protein conjugate comprising a capsular polysaccharide prepared by the method of  claim 37 . 
     
     
         40 . An immunogenic composition comprising the immunogenic polysaccharide-protein conjugate of  claim 39 . 
     
     
         41 . An immunogenic composition comprising polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group  B. streptococcus  (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII, and IX. 
     
     
         42 . An immunogenic composition comprising polysaccharide-protein conjugates comprising at least four GBS capsular polysaccharide serotypes selected from the group consisting of Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX. 
     
     
         43 . The immunogenic composition of  claim 42 , wherein the composition comprises GBS capsular polysaccharide serotype V. 
     
     
         44 . The immunogenic composition of  claim 43 , wherein the composition does not have immune interference.