Vaccination remains the most effective strategy for prevention of infectious diseases. In recent years, considerable advances have also been made in the development of therapeutic cancer vaccines. The most effective vaccines elicit both innate and adaptive immune responses, resulting in a sustained stimulation of cellular and humoral immune systems, which is particularly significant for therapeutic vaccines. The project, of which the research in this master's thesis is a part, is aimed at developing a vaccine delivery system based on filamentous bacteriophage, whereby the virion serves as an antigen-carrying scaffold with intrinsic adjuvant properties. Given the pivotal role of dendritic cells in initiating adaptive immune responses, targeting these cells can improve T-cell responses. In this master's thesis we focused on selecting nanobodies as Clec9A receptor ligands to be used for targeted delivery of filamentous bacteriophages to dendritic cells. Clec9A is a dendritic cell C-type lectin like receptor that mediates antigen uptake and its targeting to early phagosomal compartments. Targeting Clec9A represents a strategy to enhance cross-presentation leading to induction of the cytotoxic T lymphocyte response, but it also facilitates a robust humoral response characterised by follicular helper T cell generation. Due to size limitations of direct ligand display on bacteriophage capside protein pIII, we employed an indirect display method utilising the SnoopTag/SnoopCatcher autocatalytic split protein conjugation system. Six nanobodies (N1-N6) were expressed as SnoopCatcher-fused proteins in the periplasm of Escherichia coli cells. Bacteriophages f88KE, capable of dual display on capsid proteins pIII and pVIII, engineered to display SnoopTag peptides on pIII, were also expressed. The formation of a stable isopeptide bond between SnoopTag and SnoopCatcher-nanobody fusion protein was confirmed by western blotting. The binding affinity of nanobody-phage conjugates to recombinant mouse Clec9A ectodomain was assessed using a phage enzyme-linked immunosorbent assay. Aditionally, the interaction between unconjugated nanobodies and Clec9A was evaluated using enzyme-linked immunosorbent assay and biolayer interferometry. Nanobody N2 exhibited highest affinity to Clec9A, while N1 and N3 showed moderate binding. The other nanobodies showed no significant affinity. N2 will be employed in future studies to investigate the internalisation of bacteriophage conjugates by dendritic cells. Further work will also investigate the induction of the immune responses following administration of antigen-carrying bacteriophages targeting dendritic cells.
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