Development and applications of filamentous phage-derived particles in immunotherapy and diagnostics : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University (Manawatū), New Zealand

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Massey University
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Most vaccines that are currently in clinical use induce antibody-mediated responses. However, for many infectious diseases, T cells are an essential part of naturally acquired protective immune responses. T cell-inducing vaccines, such as the one developed in this research, could additionally be used for treatments in cancer or chronic viral infections. One way to target the immune cells and stimulate their responses is to use filamentous phage particles. Filamentous phages (Ff) are ssDNA viruses that infect Escherichia coli, which have been adapted and used extensively in phage display technology and nanotechnology. In this research, a filamentous phage (Ff)-based vaccine carrier was constructed to allow the tuneable display of non-protein immune adjuvant molecules (BODIPY-α-GalCer) and antigenic peptides (OVA; MHC I + MHC II) on the same particle. For the first time, azide groups were incorporated into the recombinant pVIII phage coat proteins that expressed recombinant peptides. Azide groups were subsequently used to attach fluorescently labelled adjuvant molecules, which were successfully presented to NKT cells in vivo. Additionally, high induction of in vitro proliferation of OVA-specific CD8+ T-cells was achieved. However, the Ff-derived particle use outside the laboratory is hindered because they are genetically modified viruses, which in addition, carry antibiotic resistance genes that can be horizontally transferred to gut bacteria. These limitations were overcome by developing a system for efficient production of non-replicating, controllable-length protein-DNA nanorods, derived from Ff, named BSFnano (~100 × 6 nm). In this research, functionalised BSFnano particles were constructed, and their application in diagnostics was tested in a proof of concept dipstick assay for detection of a soluble analyte (fibronectin). For the first time, an ultrasensitive dipstick assay was achieved with Ff-derived nanorods, detecting the test-analyte at a concentration of as low as 0.04 pg/μL, equivalent to only 100,000 molecules/μL. Overall, while the phage-based vaccine produced in this research elicited CD8+ T-cell responses in vitro, but not in vivo, the Ff-derived nanorods were successfully functionalised and tested in lateral flow immunoassay, with promising implications for use in point of care diagnostics.
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Bacteriophages, Nanobiotechnology, Vaccines, T cells, Point-of-care testing