Synthesis of Galα(1,3)Galβ(1,4)GlcNAcα-, Galβ(1,4)GlcNAcα- and GlcNAc-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Gal alpha(1,3)Gal beta(1,4)GlcNAc alpha, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-alpha-Gal antibodies (Abs) in infected individuals. Here, we have efficiently synthesized the mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine serum albumin (BSA). Chemiluminescent-enzyme-linked immunosorbent assay revealed that Gal alpha(1,3)Gal beta(1,4)GlcNAc alpha-BSA is recognized by purified anti-alpha-Gal Abs from chronic ChD patients similar to 230-fold more strongly than by anti-alpha-Gal Abs from sera of healthy individuals (NHS anti-alpha-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Gal alpha(1,3)Gal beta(1,4) GlcNAc alpha similar to 20-fold more strongly than pooled NHS. In contrast, the underlying disaccharide Gal beta(1,4)GlcNAc alpha and the monosaccharide GlcNAc alpha or GlcNAc beta conjugated to BSA are poorly or not recognized by purified anti-alpha-Gal Abs or sera from Chagasic patients or healthy individuals. Our results highlight the importance of the terminal Gal alpha moiety for recognition by Ch anti-alpha-Gal Abs and the lack of Abs against nonself Gal beta(1,4) GlcNAc alpha and GlcNAc alpha glycotopes. The substantial difference in binding of Ch vs. NHS anti-alpha-Gal Abs to Gal alpha(1,3)Gal beta(1,4)GlcNAc alpha-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Gal alpha(1,3)Gal beta(1,4)GlcNAc alpha-BSA NGP was then used to immunize alpha 1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Gal alpha(1,3)Gal beta(1,4)GlcNAc alpha glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD.