Specific Recognition of beta-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients' blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method's limit of detection. Previously, we focused on the detection of specific anti-alpha-galactopyranosyl (alpha-Gal) antibodies in chronic CD (CCD) patients elicited by alpha-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal beta-galactofuranosyl (beta-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients' sera react specifically with synthetic beta-Galf -containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galf beta 1,3Manp alpha-(CH2)3SH (glycan G29SH) and Galf beta 1,3Manp alpha 1,2-[Galf beta 1,3]Manp alpha-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galf beta 1,3Manp alpha 1,2-[Galf beta 1,3]Manp alpha is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.