Structural and biological insights into novel adenovirus-based platforms for therapeutic applications.

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To develop safe and effective viral platforms, it is necessary to understand the structural and molecular mechanisms by which viruses mediate cell entry and trafficking through the cell to the nucleus, where both trans- and viral gene expression occur. Furthermore, understanding the biological impact on the cell of being transduced by a therapeutic vector will be critical in ensuring that developed platforms are safe and effective for patient translation. The best studied adenovirus platform is based on human Ad5, a species C adenovirus. Whilst this virus is well understood and useful experimentally for overexpression studies, the limitations of this platform for clinical applications are well established and include the high potential for off target uptake - primarily by the liver, and high rates of pre-existing immunity against Ad5 in the general population. Generation of new platforms based on alterative serotypes with naturally lower rates of immunity in the community and lower off target uptake is therefore highly desirable. This project will therefore generate new insights into structure and biology of novel adenoviruses derived from species D and B, where numerous platforms have been developed in house and are ready for assessment. The project will be broadly split into the following general areas: Structural insights into viral structure and cellular binding mediated by the fiber knob domain. The major cellular attachment protein, the fiber knob domain, can be generated recombinantly in bacteria and purified. Recombinant fiber knob proteins from understudied adenoviral species will be generated, purified and seeded for crystallization. Once crystallized, the structure of the trimer will be solved and used for predictive modelling in complex with known adenoviral receptors. Furthermore, where possible we will attempt to co-crystallize recombinant knob protein in complex with the receptors, and binding affinities will be gauged using in silico assays such as surface plasmon resonance. These studies will be complemented by full structural insights of the virus using cryo electron microscopy. Biological insights into cellular uptake. To complement the first area, binding and entry of viruses harboring novel knob proteins will be assessed biologically. Recombinant knob protein will be used for IC50 experiments using cell lines overexpressing known adenoviral receptors (CHO-CAR, CHO-BC1, CHO-DSG2). These studies will be complemented by studies evaluating the uptake of recombinant fiber knob protein and the trafficking in endosomes using immunofluorescence (the recombinant knob protein contains a HIS tag which can be detected by IF). Ad5 based knob pseudotypes will be generated, and their ability to transduce cells bearing known adenovirus receptors will be assessed. Similarly, where available, assays will be performed using whole serotype vectors. To assess the viral transcriptome in infected cells, either normal fibroblasts or transformed cells will be infected with wt viral serotypes or the non-replicating viral vectors developed from them. The transcriptome will be analyzed using Nanopore, in collaboration with Dr David Matthews (Bristol) and viral transcripts quantified. These studies will be complemented by in vitro cell killing assays in normal and transformed cells. In vivo assessment as vaccine vectors. Finally, we will assess the potential of vectorized viral platforms as vaccines. Mice will be inoculated either intramuscularly or intranasally with GFP expressing vectors, and T-cell and antibody responses against GFP will be assessed. Collectively these data will provide critical in vitro and in vivo insights into novel and patentable viral platforms for therapeutic applications.