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 .- 34 . (canceled) 
     
     
         35 . A method of making an immunogenic polysaccharide-protein conjugate comprising the steps of:
 (a) reacting a GBS capsular polysaccharide with an oxidizing agent resulting in an activated polysaccharide; and   (b) reacting the activated polysaccharide with a carrier protein resulting in a polysaccharide-protein conjugate.   
     
     
         36 .- 44 . (canceled) 
     
     
         45 . The method of  claim 35 , wherein step (b) is carried out in a polar aprotic solvent. 
     
     
         46 . The method of  claim 45 , wherein the solvent is selected from the group consisting of dimethylsulfoxide (DMSO), sulfolane, dimethylformamide (DMF), and hexamethylphosporamide (HMPA). 
     
     
         47 . The method of  claim 46 , wherein the solvent is dimethylsulfoxide (DMSO). 
     
     
         48 . The method of  claim 35 , wherein the oxidizing agent is a periodate. 
     
     
         49 . The method of  claim 48 , wherein the periodate is sodium periodate. 
     
     
         50 . The method of  claim 35 , 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. 
     
     
         51 . The method of  claim 35 , wherein the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). 
     
     
         52 . The method of  claim 51 , wherein N-chlorosuccinimide (NCS) is a cooxidant. 
     
     
         53 . The method of  claim 35 , wherein step (a) further comprises quenching the oxidation reaction by addition of a quenching agent. 
     
     
         54 . The method of  claim 35 , wherein the method further comprises the step of lyophilizing the activated polysaccharide. 
     
     
         55 . The method of  claim 54 , wherein the activated polysaccharide is lyophilized in the presence of a saccharide selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit. 
     
     
         56 . The method of  claim 35 , wherein step (b) comprises:
 (1) compounding the activated polysaccharide with a carrier protein, and   (2) reacting the compounded activated polysaccharide and carrier protein with a reducing agent to form a GBS capsular polysaccharide-carrier protein conjugate.   
     
     
         57 . The method of  claim 56 , wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride, sodium triacetoxyborohydride, sodium and zinc borohydride in the presence of Bronsted or Lewis acids, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i PrN—BH 3 , benzylamine-BH 3  or 5-ethyl-2-methylpyridine borane (PEMB). 
     
     
         58 . The method of  claim 57 , wherein the reducing agent is sodium cyanoborohydride. 
     
     
         59 . The method of  claim 35 , wherein the method further comprises a step (step (c)) of capping unreacted aldehyde by addition of a borohydride. 
     
     
         60 . The method of  claim 59 , wherein the borohydride is selected from the group consisting of sodium borohydride (NaBH 4 ), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, and magnesium borohydride. 
     
     
         61 . The method of  claim 60 , wherein the borohydride is sodium borohydride (NaBH 4 ). 
     
     
         62 . The method of  claim 35 , wherein the method further comprises the step of purifying the polysaccharide-protein conjugate. 
     
     
         63 . 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.