Reversed Immunoglycomics Identifies Alpha-Galactosyl-Bearing Glycotopes Specific For Leishmania Major Infection

All healthy humans have high levels of natural anti-alpha-Galactosyl (alpha-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of alpha-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in alpha-Gal-based vaccines. Patients with certain protozoan infections have specific anti-alpha-Gal antibodies, elicited against parasite-derived alpha-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Gal alpha 1,3Gal beta 1,4Glc-NAc alpha, expressed by Trypanosoma cruzi. The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific alpha-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major, the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic alpha-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Galp alpha 1,3Galf beta and Galp alpha 1,3Galf beta 1,3-Manpa glycotopes as diagnostic biomarkers for L. major-caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica-caused OWCL. These glycotopes could prove useful in the development of rapid alpha-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic alpha-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-alpha-Gal antibodies.