Synthetic antigens based on the glycan structure of mucins from the infective forms of Trypanosoma cruzi

LOPEZ, LAURA ROSANA; DE LEDERKREMER, ROSA M.; DUCREY, IVANA; GIORGI, M. EUGENIA

Buenos Aires, CABA

Simposio; Exploring the fronters of chemistry: Challenges for 21st century; 2019

UBA

The immunodominant glycotope α-Galp-(1→3)-β-Galp-(1→4)-GlcNAc, also known as α-Gal, expressed in the mucins of the infective trypomastigote stage of Trypanosoma cruzi has been proposed for multiple clinical applications, from xenotransplantation or cancer vaccination to serodiagnosis of Chagas disease. Regarding the latter, however, methodological limitations have precluded its consistent clinical use. It was previously shown that the trisaccharide analogue to α-Gal, with Glc in the reducing end instead of GlcNAc, was as efficient as the natural trisaccharide for recognition of antibodies to α-Gal elicited during T. cruzi infections. We describe here convenient syntheses of α-Galp-(1→3)-β-Galp and α-Galp-(1→3)-β-Galp-(1→4)-Glcp, both functionalized as the 6-aminohexyl glycosides, and their conjugation to BSA. For the synthesis of the trisaccharide a lactose derivative, which already has the β-Galp-(1→4)-β-Glcp motif, was used as starting material. For conjugation, the squarate method was chosen, which allowed the direct derivatization of the lysine amino groups of BSA by the carbohydrate moieties. The synthesized neoglycoconjugates were structurally characterized by biochemical and mass spectrometry studies and antigenically validated by conventional ELISA immunoassays. Both α-Galp-(1→3)-β-Galp-(1→4)-Glcp and α-Galp-(1→3)-β-Galp were specifically recognized by serum samples of T. cruzi-infected patients. Moreover, competition assays allowed us to map the disaccharide α-Galp-(1→3)-β-Galp as the glycotope recognized by anti-a-Gal antibodies, thereby supporting the ?antigenic mimicry? between α-Galp-(1→3)-β-Galp-(1→4)-Glcp and the natural α-Gal structure. The α-Galp-(1→3)-β-Galp disaccharide was next conjugated by the squarate method to immunodominant peptides present in T. cruzi recognized antigens. Further immunoassays using unconjugated peptides and 6-aminohexyl α-Galp-(1→3)-β-Galp as controls indicated that it is possible to develop bivalent serological reagents, able to display peptidic- and carbohydrate-based epitopes. Overall, these results indicate that our neo-glycoconjugates provide suitable, cost-effective and much needed tools for the improvement of currently used Chagas disease diagnostic applications.