Synthesis and preclinical evaluation of glycoconjugate vaccines against group B Streptococcus types VI and VIII.

Group B Streptococcus (GBS) types VI and VIII are prevalent among serotypes isolated from pregnant women in Japan. Maternal vaccination with a safe and effective GBS vaccine has been proposed as a rational approach to prevent neonatal GBS disease. Because antibody specific for the capsular polysaccharide (CPS) antigens of GBS is protective, vaccines were developed with purified type VI and VIII CPS coupled to tetanus toxoid. In rabbits the newly synthesized conjugate vaccines elicited high-titered, type-specific antibody that was opsonically active in vitro. Moreover, litters born to mice actively vaccinated with the conjugate vaccines, in contrast to uncoupled CPS or saline, were protected against an ordinarily lethal challenge of GBS of homologous serotype. GBS types VI and VIII conjugate vaccines of the design presented may be important components of a multivalent GBS vaccine for use in regions where these serotypes predominate.

Bacteroides fragilis strains express multiple capsular polysaccharides.

Previous studies by our group have demonstrated that the capsule of Bacteroides fragilis type strain NCTC 9343 consists of two chemically distinct polysaccharides, designated PS A and PS B. These polysaccharides can be isolated as an aggregate from the surface of the organism and give a complex multiprecipitin profile when they are reacted with homologous antiserum in an immunoelectrophoresis assay. Following structural analysis of PS A and PS B, we have determined that the complex precipitin profile is formed as a result of the differing electrophoretic and antigenic properties associated with each of these polymers. Presently, we have examined the capsular polysaccharides of 13 other strains of B. fragilis according to methods used for the prototype strain. The capsules of these strains were extracted, partially purified, and analyzed by immunoelectrophoresis at pH 7.3. Following reaction with homologous polyclonal antisera, each of the capsular preparations tested yielded a complex precipitin profile similar to that of the prototype strain. When reacted by immunoelectrophoresis with polyclonal antiserum to 9343 or with monoclonal antibodies to PS A and PS B, these capsular preparations appeared to be antigenically diverse; some preparations (50%) showed complete or partial cross-reaction. These results suggest that the dual polysaccharide motif seen with the prototype strain is a common feature of B. fragilis strains. In addition, the antigenic heterogeneity of B. fragilis capsular polysaccharides could be used for the development of a serological typing scheme.

Variations of Bacteroides fragilis with in vitro passage: presence of an outer membrane-associated glycan and loss of capsular antigen.

The amount of capsular antigen produced by Bacteroides fragilis appears to decrease significantly with in vitro passage on blood agar plates of strain 23745. This decrease in the quantity of isolable capsule is associated with the emergence of a small transparent colony type variant. The small colony type strain also has a glycogenlike material associated with its outer membrane. This glycogen is found intracellularly but is not associated with the outer membrane on the mucoid colony variant that predominates after passage in animals--the large colony type. No concomitant alteration in outer membrane proteins or lipopolysaccharides is associated with this colonial transformation. Electron micrographs, with either ruthenium red staining or indirect ferritin antibody staining of the capsular antigen, confirm the relative loss of capsule with in vitro passage. Thus, care must be taken during in vitro studies of the antigenic structure of B. fragilis to define the degree of passage of the strain.

Isolation and identification of encapsulated strains of Bacteroides fragilis.

One hundred three clinical isolates of Bacteroides fragilis were identified during a two-year period. Most of these isolates were strains of B. fragilis subspecies fragilis, which constitutes a minor component of the fecal flora in comparison with the other subspecies of B. fragilis. By use of several techniques for demonstration of capsules, it was found that only B. fragilis strains classified as subspecies fragilis were encapsulated. An indirect immunofluorescence assay was developed for identification of clinical isolates possessing capsular material that was immunologically similar to that found in the reference strain of B. fragilis subspecies fragilis. All strains examined that were classified as subspecies fragilis were positive in this assay for the capsular material, whereas strains of the other subspecies were negative. This tests represents a rapid and sensitive means of identifying the most prevalent anaerobic gram-negative bacillus involved in human infections. The capsular polysaccharide of B. fragilis subspecies fragilis is a unique factor associated with the predominant subspecies of B. fragilis isolated from clinical material.