Synthesis and evaluation of neo-glycoconjugates as tools for the serological diagnosis of Chagas disease.

DUCREY, IVANA; LOPEZ, ROSANA; GONZALEZ-SALAS, DIEGO ; MARINO, CARLA; BALOUZ, VIRGINIA; MELGAREJO, LINDA TORO; LEDERKREMER RM; CÁMARA, MARÍA DE LOS MILAGROS; GIORGI M.E.; AGÜERO, F; BUSCAGLIA, CARLOS A.

Mar de Plata

Congreso; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; 2019

Sociedad Argentina de Protozoología

The immunodominant glycotope -Galp-(1→3)--Galp-(1→4)-GlcNAc (also known as-Gal), expressed in the mucins of the infective trypomastigote stage of Trypanosoma cruzi has been proposed for multiple clinical applications, from xenotransplantation or cancer vaccinology to serodiagnosis of protozoan caused diseases, including Chagas disease. Regarding the latter, however, methodological limitations has precluded its consistent use in the clinics. It was previously shown that the analogue trisaccharide, with Glc in the reducing end instead of GlcNAc, was as efficient as the natural trisaccharide for recognition of antibodies to -Gal elicited during T. cruzi infections. We describe here the synthesis of α-Galp-(1→3)-β-Galp-(1→4)-Glcp and α-Galp-(1→3)-β-Galp, both functionalized as the 6-aminohexyl glycoside, and their conjugation to BSA. For the synthesis of the trisaccharide a lactose derivative, which already had the β-Galp-(1→4)--Glcp motif, was used as starting material. For conjugation, the squarate method was chosen, which allowed the direct derivatization of the lysines 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-Gal as the glycotope recognized by anti--Gal antibodies, thereby supporting the ?antigenic mimicry? between α-Galp-(1→3)-β-Galp-(1→4)-Glcp and the natural -Gal structure. The α-Galp-(1→3)-β-Gal disaccharide was next conjugated to immunodominant peptides present in T. cruzi recognized antigens. Further immunoassays using unconjugated peptides and α-Galp-(1→3)-β-Gal structures 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