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Biochemical characterisation of a novel vaccine candidate against Trypanosoma vivax, the cell surface glycoprotein IFX https://doi.org/10.17863/CAM.117395 Mackenzie, Craig Luisi, Ben Carrington, Mark Paszkowski, Uta Wright, Gavin This thesis reports the expression, purification, biochemical characterisation and antibody recognition of a recently discovered cell surface glycoprotein and vaccine candidate, Invariant Flagellum Antigen from Trypanosoma vivax (IFX) from the kinetoplastid parasite Trypanosoma vivax. African trypanosomes are unicellular protozoan parasites and pathogens from the family Trypanosomidae, able to infect nearly all mammalian species. African animal trypanosomiasis (AAT) affects both domesticated and wild animals and is a pressing issue for livestock farmers in Sub- Saharan Africa and South America. There is a need for effective prophylaxis and improved therapy of this devastating illness, and recently, antibodies against IFX have been shown to confer protection against the disease in a mouse model system. Passive transfer experiments administering those anti-IFX monoclonal antibodies to naive mice challenged with T. vivax previously revealed that some were protective and some were not. This thesis project sought to understand how the epitopes are recognised at the structural level and why there is not a straightforward correlation between antibody affinity and protective efficacy. To address this, the stability and behaviour of IFX from T. vivax was assessed biophysically and interactions examined with specific anti-IFX monoclonal antibodies which had been previously raised in IFX-immunised mice where sterile immunity was observed upon challenge with T. vivax. Mapping of epitopes for the protective antibodies on IFX during an IFX-induced adaptive immune response may help to optimise an effective vaccine targeted to those regions of IFX. Furthermore, such a subunit protein vaccine must be stable and the behaviour of the protein constituent understood biophysically. The first results chapter reports the expression and purification of recombinant IFX, testing different constructs in bacterial, insect and mammalian systems. Structural predictions for IFX alone, its self-association and glycosylation pattern are presented and evaluated. Issues with protein stability were overcome after extensive exploration, leading to a construct with the IFX extracellular domain fused to a short EPEA tag at the C-terminus. Cell surface expression of IFX heterologously on insect cells was also explored, with a view to optimising this system for immunological cell-based experiments in future. Structurally denatured material from bacterial expression (Escherichia coli ) was used to map linear epitopes on IFX for purified anti-IFX murine monoclonal antibodies. The second results chapter presents biochemical and biophysical analyses of recombinant IFX and preliminary efforts for electron microscopy and crystallisation. Circular dichroism established that the extracellular domain of IFX is mainly alpha helical in structure composition, which agrees with predictions. Mass photometry analyses indicate that the IFX extracellular domain exists mainly as a mixture of monomers and dimers, but also forms some larger oligomers. The multiple IFX glycoforms visible by denaturing gel electrophoresis were verified to be IFX by mass spectrometry. Sedimentation velocity analysis demonstrated that the total fraction of IFX extracellular domain was forming multiple stable oligomers, but that the isolated lower glycoform displayed fast reversible self-association. The third results chapter examined the interaction of IFX with anti-IFX monoclonal antibodies of relevance to an IFX-induced adaptive immune response in mice. It was established that one of the protective anti-IFX monoclonal antibodies and two of the non-protective ones recognise linear epitopes on IFX. In contrast, another of the protective anti-IFX monoclonal antibodies is more likely to recognise a conformational epitope. With this recognition established and knowing that the linear epitopes map broadly to an N-terminal region on IFX, constructs were designed with short N-terminal IFX fragments to map more finely the antibody recognition sites. Expressing these constructs in E. coli and monitoring detection by western blot, it was established that the non-protective antibody binds closer to the N-terminus of IFX than the protective counterpart. Complement recruitment accounts for a large component of IFX-elicited immunity in mice, but IFX is a bovine vaccine candidate, so an assay was designed to test complement C1q recruitment to specific bovine IgG isotypes to help fine tune a bovinised IFX vaccine in future. Considering that certain trypanosome infections can be chronic, and that studies on kinetoplastids have been largely focused on pathogenic trypanosomes (despite large groups of kinetoplastids being non-pathogenic), a separate symbiosis study was included in the thesis on a beneficial symbiosis in nature. Although this work was completely separate to the studies on IFX, it was a useful bridge to acknowledging mutualistic symbioses in nature alongside the main parasitology study. The study focused on the signalling specificity of an intracellular receptor, Dwarf 14-Like (D14L), essential for arbuscular mycorrhizal (AM) symbiosis in monocotyledonous plants and with a dual role in developmental signalling in both monocotyledonous and dicotyledonous species. It was found that the catalytic triad of D14L in Oryza sativa is required for AM symbiosis signalling. In addition, initial data from primary transformants indicated that D14L developmental signalling is conserved between dicotyledonous and monocotyledonous plant species. Given the dual role of D14L in AM symbiosis and development, constructs were designed to determine which domains of D14L are involved in AM symbiosis specifically. Overall, this thesis has established an expression and purification system for IFX from Trypanosoma vivax that has yielded helpful biochemical and biophysical information, but also provides a foundation to support structural studies in future. By narrowing down the broad region for the linear epitope for the protective antibody on IFX and establishing that a second antibody has a conformational epitope, the information obtained will inform fine tuning of IFX as a vaccine and works towards understanding how these two antibodies neutralise a Trypanosoma vivax infection via targeting IFX. University of Cambridge 2024-10-31 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/382716 https://www.repository.cam.ac.uk/bitstreams/ac7d36c1-8cc1-4ac9-a2f0-d38918e88e5c/download 99a27c0283cec668a89446c75bbe2c0e https://www.repository.cam.ac.uk/bitstreams/4cf5bddc-f2ec-4b25-a15f-943926ea1b23/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved African trypanosomiasis Antibody-antigen interactions Arbuscular mycorrhizal fungi Cell surface glycoprotein Chinese Hamster Ovary mammalian cell expression system Epitope characterisation Escherichia coli bacterial cell expression system IFX-elicited immunity Mutualistic symbiosis Oligomerisation Protein folding Recombinant protein expression and purification Symbiosis signalling Trichoplusia ni insect cell expression system Trypanosoma vivax Vaccine design Invariant Flagellum Antigen from Trypanosoma vivax (IFX) DWARF14-LIKE (D14L)