(539) Characterization and Functional Assessment of IB4-Fc (IgG) Fusion Proteins Targeting Trypanosoma cruzi Surface Glycoconjugates

Introduction/Rationale: Glycoconjugates on the surface of Trypanosoma cruzi are critical for parasite survival and host–pathogen interactions. α-galactose–containing glycotopes are abundant on infective trypomastigote surface molecules and absent from human glycoconjugates, rendering them highly immunogenic. These structures elicit lytic antibody responses and have been explored as immunogens in experimental vaccine studies. α-galactose–containing glycoconjugates also contribute to host cell invasion. Lectins are glycan-binding proteins, and fusion to Fc domains enables coupling of glycan recognition with innate immune effector mechanisms. Here, we performed an expanded characterization of IB4-Fc fusion proteins targeting terminal α-galactose–containing glycotopes on T. cruzi trypomastigotes.

Methods: IB4-Fc fusion proteins incorporating different IgG isotypes were expressed in CHO cells and purified by affinity chromatography. Protein integrity was assessed by SDS-PAGE. Binding was evaluated using synthetic α-galactose–containing glycans and T. cruzi trypomastigotes in dose-dependent assays. Parasite surface binding was examined by fluorescence microscopy and flow cytometry. Functional activity was assessed in vitro using macrophage assays to evaluate parasite uptake and damage.

Results: B4-Fc fusion proteins showed appropriate biochemical integrity and dose-dependent binding to α-galactose–containing glycans and intact parasites. Fluorescence microscopy revealed focal binding patterns on the parasite surface, as assessed by flow cytometry. IB4-Fc fusion proteins enhanced macrophage-mediated parasite uptake and promoted parasite damage in vitro. Glycoimmunoarray profiling to define α-Gal–based glycotopes recognized by IB4-Fc is ongoing.

Conclusion: IB4-Fc fusion proteins recognize α-galactose–containing glycotopes on T. cruzi and engage innate immune responses in vitro. Ongoing glycotope mapping will further define the molecular basis of IB4-Fc recognition and support glycotope-directed strategies for Chagas disease.