Oligosaccharide fragments of the type III group B streptococcal polysaccharide derived from S. pneumoniae type 14 capsular polysaccharide by a chemoenzymatic method.

Partial N-deacetylation fo the GlcNAc residues in S. pneumoniae type 14 capsular polysaccharide (Pn14-PS) backbone was achieved by treatment with base, and the product was subsequently enzymatically sialylated at the 3-O-positions of the terminal galactose residues. The resultant, partially N-deacetylated type III Group B streptococcus capsular polysaccharide (GBSIII-PS) was subjected to nitrous acid deamination, which resulted in the degradation of GBSIII-PS polysaccharide into oligosaccharides containing increasing numbers of the identical repeating units. The oligosaccharides were then separated by passage through a Superdex 30 column and characterized by ESIMS and NMR spectroscopic analysis.

Streptococcus pneumoniae type 14 polysaccharide-conjugate vaccines: length stabilization of opsonophagocytic conformational polysaccharide epitopes.

A simple and convenient method was developed for the preparation of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS)-tetanus toxoid (TT) conjugate vaccines, using terminally linked Pn14PS fragments of different lengths. Native Pn14PS was simultaneously depolymerized and activated for conjugation by partial N-deacetylation followed by nitrous acid deamination which yielded fragments (1.4 to 150.0 kDa) having a free aldehyde at the reducing end. These were then conjugated to TT through their terminal aldehydic groups, using the reductive amination procedure. All of the above conjugates, when injected in rabbits, induced anti-Pn14PS antibodies, whereas the native Pn14PS did not. The amounts of anti-Pn14PS antibodies elicited by these conjugates, as determined by enzyme-linked immunosorbent assay, followed a trend with conjugates containing the highest-molecular-weight Pn14PS eliciting the highest titers. The same trend was also observed in the ability of the antibodies to opsonize and kill live type 14 pneumococci, although the increase in opsonophagocytic activity was more pronounced and did not correlate linearly with increases in antibody titer. Competitive inhibition of the binding of different conjugate antisera to the native Pn14PS, using Pn14PS fragments as inhibitors, established that the conjugates induced antibodies with specificities for different lengths of Pn14PS beginning at 2 repeating units (RU). It was also established, both immunologically and antigenically, that at least 4 RU of Pn14PS were required to form an extended conformational epitope and that approximately 22 RU of Pn14PS were required to duplicate the same epitope on the same saccharide chain. The conformational epitope was found to be essential for the induction of antibodies with high opsonophagocytic activity and that augmentation of opsonophagocytic activity was also dependent on further chain extension.

The synthesis of Streptococcus pneumoniae polysaccharide-tetanus toxoid conjugates and the effect of chain length on immunogenicity.

To study the relationship between length of pneumococcal polysaccharide and immunologic performance in rabbits we took well defined fragments of the capsular polysaccharides of S. pneumoniae types 3, 6A, 18C, 19F and 23F and pneumococcal C-polysaccharide and linked them terminally by reductive amination to tetanus toxoid. Contrary to other reports we found little variation in antibody titers with increasing length. In general the opsonophagocytic titers determined using activated HL60 cells and rabbit peritoneal cells correlated well with the antibody titers except for that of type 3, which despite the presence of high polysaccharide antibody titers gave unexpectedly low opsonophagocytic titers. The C-polysaccharide-conjugate was also immunogenic when injected in both rabbits and mice but gave low opsonophagocytic titers. It was demonstrated that opsonophagocytosis was solely dependent on the presence of phosphoryl choline-specific antibody and that the induction of these antibodies was species dependent.

Inhibition of influenza A virus hemagglutinin and induction of interferon by synthetic sialylated glycoconjugates.

Multivalent forms of neoglycoproteins and polyacrylamides containing sialic acid were prepared and shown to be potent inhibitors of influenza A virus (H3N2) hemagglutinin with chick red blood cells. The synthetic sialylated glycoconjugates, although they were neuraminidase substrates, did not suppress viral neuraminidase and did not reduce infectivities in chick embryos. The copolyacrylamide conjugate containing a spacer group of approximately 11 A (1 A = 0.1 nm) between the polymer backbone and the sialic acid residues was the best hemagglutinin inhibitor. Moreover, it exhibited promising interferon-inducing properties.