b-D-Gal-(1→4)-b-D-Glc-(1→6)-[b-D-Gal-(1→4)]-D-GlcNAc linked to an immunogenic protein represents the smallest structure that evokes antibody responses against Streptococcus pneumoniae type 14 (SP14). Recent studies1 have shown that some zwitterionic capsular polysaccharides (CPS) have an intrinsic ability to induce specific T cell-dependent immune responses. On this basis, zwitterionic CPS 1 and 2 were ex novo synthesized to understand whether the stimulation of T cell responses against SP14 is possible in absence of conjugation with immunogenic proteins.
Two disaccharide acceptors 4a and 4b were used to introduce the cation centre in position 6’(4a) or on the aglycone moiety (4b).
The same lacturonic donor 3 carrying the anionic component was used for glycosylating 4a and 4b to give both zwitterionic tetrasaccharides.
The zwitterionic tetrasaccharide 1, its anionic and cationic models, and the neutral methyl glycoside analogue, were biologically tested for their biocompatibility (MTT test) and their ability to compete with natural SP14 CPS for binding to a specific antibody (ELISA). No compounds induced cytotoxicity on macrophage cells (RAW 264.7) at any concentrations tested (10-2-102 mg/ml) after 12-24 h. All analogues were recognized by a specific SP14 antibody with similar affinities (IC50=10-5 mg/ml), but showed a lower efficacy than the native SP-14 CPS (30% vs 100% of inhibition at 10-1 mg/ml). To rule out the possibility that the analogues bound nonspecifically to the plate surface, we performed control experiments by coating the ELISA plates with colominic acid, a polysaccharide from Escherichia coli. Results indicate that no nonspecific binding occurs under these experimental conditions.
In order to evaluate the effect of a different location of the cationic charge, the same biological tests are currently in progress with the zwitterionic tetrasaccharide 2.
1McLoughlin, R.M.; Kasper D.L. Microbial Glycobiology: Structures, Relevance and application 2009, 49, 957.
