INTRODUCTION: African trypanosomosis is a disease of medical and veterinary importance threatening both humans and animals in Sub-Saharan Africa. Currently, there are no anti-disease vaccines available and disease control relies heavily on the treatment with trypanocidal drugs. Therefore, targeting a trypanolytic/toxic molecule to a parasite surface molecule or receptor is ideal for increasing the drug targeting efficiency. In this study, we opted to use the trypanosomal transferrin receptor (TfR), a GPI-surface linked heterodimeric protein (ESAG6 and 7) bearing no similarity to the mammalian transferrin receptor involved in iron-uptake, as a target molecule. We propose to use a Nanobody technology® platform to generate and characterize Nanobodies® against the bloodstream trypanosomal transferrin receptor (ESAG6) as a tool to target trypanosomes. METHODOLOGY: Following immunization of a llama with recombinant ESAG6 and selection against the ESAG6 via phage-display technology, three monovalent anti-ESAG6 Nanobodies® were selected. These were initially characterized in silico and after purification, via thermostability assays. The anti-ESAG6 Nanobodies® were subsequently tested in an Enzyme Linked Immunosorbent assay (ELISA) using whole cell lysate from different purified bloodstream-derived trypanosome species. In addition, Flow cytometry was performed with Alexa Fluor® 647 labelled anti-ESAG6 Nanobodies® on purified bloodstream-derived and blood-borne monomorphic trypanosomes. RESULTS: The Nanobody® characterization assays revealed stable proteins with an expected molecular weight of 14 kDa and good thermal stability. ELISA assays indicated specific binding to T. brucei - derived lysate with little cross-reactivity to non - T. brucei related trypanosomal lysates. Flow cytometry analysis confirmed that one Alexa Fluor® 647 anti-ESAG6 Nanobody® could detect and bind to its antigen on intact monomorphic T. b. brucei Antat 1.1 parasites. In addition, a stronger signal was obtained on fixed and permeabilized parasites. CONCLUSIONS: The available anti-ESAG6 Nanobodies® can bind their antigen in parasite lysate as well as on intact parasites. Moreover, binding of anti - ESAG6 Nanobodies® on fixed and permeabilized parasites indicate that the majority of TfR molecules are present intracellularly. Collectively, these results suggest that anti - TfR Nanobodies® might have diagnostic and therapeutic (targeting) potential.
5 mei 2017
Annual scientific meeting of the Belgian Society of Parasitology and Protistology 5th May 2017 VUB, Etterbeek, Brussels, Belgium