Recent efforts toward developing vaccines against group B streptococci (GBS) have focused on increasing the immunogenicity of GBS polysaccharides by conjugation to carrier proteins. However, partial depolymerization of GBS polysaccharides for the production of vaccines is a difficult task because of their acid-labile, antigenically critical sialic acids. Here we report a method for the partial depolymerization of type II and III polysaccharides by mild deaminative cleavage to antigenic fragments with reducing-terminal 2,5-anhydro-Dmannose residues. Through the free aldehydes of their newly formed end groups, the fragments were conjugated to tetanus toxoid by reductive amination. The resulting conjugates stimulated the production in animals of high-titer type II-and III-specific antibodies which induced opsonophagocytic killing of type II and III strains of group B streptococci. For the type II conjugates, immunogenicity increased as oligosaccharide size decreased, whereas for type III conjugates, the size of the oligosaccharides did not significantly influence immunogenicity. When oligosaccharides of defined size were conjugated through sialic acid residues, the resulting cross-linkages were shown to affect immunogenicity. When oligosaccharides were conjugated through terminal aldehyde groups generated by deamination, modification of the exocyclic chain of sialic acid did not influence immunogenicity.Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis and of invasive infections in nonpregnant adults with underlying illnesses (7). Although antibodies directed to the capsular polysaccharide (CPS) antigens are protective, these antigens are variably immunogenic (6). The immunogenicity of GBS CPS antigens has been increased by covalent coupling to proteins to form CPS-protein conjugate vaccines (1-5, 18, 21, 23, 25, 29). Experimental GBS type III polysaccharide (GBSP-III)-and oligosaccharide-tetanus toxoid conjugate vaccines of different designs have been developed and their immunogenicity tested in animals (21, 29). (The term "oligosaccharide" is generally used to designate carbohydrates containing between 2 and 10 monosaccharide units per molecule [12]. For convenience and consistency with related published material [12,15,23,25,29], the term oligosaccharide is used in this paper to indicate a fragment obtained by chemical or enzymatic cleavage of a native polysaccharide.) Generating GBS oligosaccharides is a difficult task because of the acid-labile, antigenically critical sialic acid residues present at the termini of the side chains of all GBS CPS serotypes. Enzymatic digestion of the type III CPS with endo-␤-galactosidase allowed the production of oligosaccharide-tetanus toxoid (TT) conjugates which proved to be immunogenic in animals (21). Unfortunately, the specificity of the endo-␤-galactosidase is restricted to type III CPS. We sought a facile chemical degradation for the type III polysaccharide (PS) and possibly those of other CPS serotypes, keeping in mind the acid labilit...

In this study, we tested the hypothesis that the immunogenicity and protective efficacy of polysaccharide-protein conjugate vaccines are influenced by three variables: (i) molecular size of the conjugate, (ii) molecular size of the polysaccharide used for conjugation, and (iii) extent of polysaccharide-to-protein cross-linking. Type III group B Streptococcus capsular polysaccharide was linked by reductive amination at multiple sites to tetanus toxoid to create a polysaccharide-protein conjugate (III-TT). A single lot of III-TT was fractionated into small, medium, and large M rpools. Whereas all three conferred protection in a maternal immunization-neonatal challenge model in mice, the smallestM r conjugate evoked less polysaccharide-specific immunoglobulin G (IgG) than the two largerM r conjugates. To test whether the molecular size of the polysaccharide used for conjugation also affected the immunogenicity of the conjugate, vaccines were synthesized using capsular polysaccharides with M rs of 38,000, 105,000, and 349,000. Polysaccharide-specific IgG responses in mice increased with the M r of the polysaccharides, and protective efficacy was lower for the smallest polysaccharide conjugate compared to the other two vaccines. Immunogenicity testing of a series of vaccines prepared with different degrees of polysaccharide-to-protein cross-linking demonstrated higher polysaccharide-specific antibody responses as the extent of cross-linking increased. However, opsonic activity was greatest in mouse antiserum raised to a moderately cross-linked conjugate, suggesting that some antibodies evoked by highly cross-linked conjugates were directed to a nonprotective epitope. We conclude that conjugate size, polysaccharide size, and degree of polysaccharide-protein cross-linking influence the immunogenicity and protective efficacy of III-TT conjugate vaccines.

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