Generation and characterization of anti-T.brucei transferrin receptor Nanobodies

Student thesis: Master's Thesis

Abstract

African Animal Trypanosomosis is a major threat to animal health and production in endemic areas. There is currently no vaccine available against the disease. Control relies on treatment of infected animals with trypanocidal drugs, of which drug resistance is a major challenge. Targeting a trypanolytic molecule or surface receptors for nutrients would be ideal in controlling parasitaemia. The bloodstream forms of trypanosomes fulfil their iron requirements by taking up host transferrin via the trypanosomal transferrin receptor, a heterodimeric protein, that bears no similarity to the mammalian transferrin receptor hence ideal target by trypanocidal molecules. This study aimed to generate and characterize high-affinity anti- T. brucei transferrin receptor Nanobodies® both as monovalent formats and half-life extended formats through fusion of the Nanobody to an anti-serum albumin Nanobody®. Three anti- T. brucei transferrin receptor Nanobody® sequences were first subjected to sequence redundancy check according to the alignment of complementarity determining region 3 (CDR3), from which one Nanobody® demonstrated high CDR3 hypervariability. In silico characterization of the Nanobodies® revealed highly stable proteins with a molecular weight of approximately 14 kDa. A ThermoFluor® assay confirmed the Nanobodies® stability with a melting temperature of approximately 70°C. Collectively, the generated Nanobodies® bound to different cell lysates as detected in an Enzyme-Linked Immunosorbent Assay. Flow cytometry analysis confirmed that the Alexa Fluor® 647 labelled Nanobodies® were able to bind to their antigen in live and intact monomorphic Trypanosoma brucei brucei parasites. The Nanobodies® specifically detected the parasites within whole blood as assayed by Flow cytometry. This indicates that they have potential for application in in vivo settings. However, generating the serum half-life time extended formats of these anti-TbTfR Nanobodies®, which might be required to increase their in vivo circulation time/efficiency, was hampered by poor recombinant protein expression and purification.
Date of Award30 Aug 2016
Original languageEnglish
Awarding Institution
  • Interuniversity Program Molecular Biology
  • Vrije Universiteit Brussel
SupervisorChristopher Kinyanjui Kariuki (Advisor), Benoit Stijlemans (Promotor) & Stefan Magez (Promotor)

Keywords

  • Nanobody®
  • trypanosomes
  • transferrin receptor
  • half-life
  • serum albumin

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