Antibody engineering and antigen discovery against Avian Paramyxovirus

Thesis

Twenty-five years since the first structure of Avian Orthoavulavirus (AOav) F protein was solved in the post-fusion conformation, the structure of the pre-fusion conformation remains elusive. AOaV is a major pathogen of poultry, especially chickens, and presents major challenges to small scale farmers and larger industry alike. Vaccination is one of the most effective ways of protecting against the pathogen. The primary antigen used, the F protein, is known to transition between 2 conformations, the metastable pre-fusion conformation and the stable post-fusion conformation. The structure of the post-fusion conformation is known, but the structure of the pre-fusion conformation is unknown.

The first aim of this project was to solve the pre-fusion structure of NDV F. This structure could then be used to develop a stabilised F antigen, which would allow for the creation of a more effective vectored-vaccine by preventing the conformational change. Additionally, epitope mapping could have been performed to allow for identification of regions on the antigen that were common to multiple genotypes of the virus. All this put together would have created a candidate antigen which could be inserted into a Turkey Herpesvirus (HVT) vectored vaccine. This prototype vaccine could then be compared with current vaccines in a series of immunisation assays and eventually, challenge assays.

The project made an initial success, with protein crystallising, and then the crystals examined with X-ray crystallography. However, the structure could not be determined from this dataset. Following this, further attempts to grow crystals were made, all without success. Thus, a pivot was made to cryo-EM, again with some initial promise. The task proved problematic due to difficulties in sample preparation and the preparation of grids for cryo-EM. Meanwhile a SAXs dataset was collected and compared with some models of the protein, generated with Alpha-Fold, and other class I fusion proteins and our own results. The cryo-EM work was interrupted by the pandemic, and due to other considerations, the attempt to insert the stabilised F protein into an HVT vector was brought forwards. This attempt failed. Furthermore, the nanobodies generated in the start of the project from expressed F protein were also cloned into suitable vectors and expressed. However, it appeared that when incubated with the F protein, the nanobodies also appeared not to bind. This impacted efforts to determine the structure of the F protein as they could have been used to stabilise the protein for crystallography or cryo-EM work. Other attempts to stabilise the F protein with additives and pegylation were also initially promising, but ultimately unsuccessful. Taken as a whole, the project provides a framework for future structural studies into a protein that remains a high priority target.

Authors: Llewellyn, MAR

• DOI: 10.5287/ora-zbxj00e6r
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Avian Orthoavulavirus; structural virology; Avian Paramyxovirus; cryo-EM; Avian Avulavirus; X-Ray crystallography
• Subjects: Avian virology; Structural studies