Topographical, mechanical, and structural investigation of virus-like particles and self-assembling protein nanoparticles

Virus-like particles (VLPs) are self-assembling protein nanocages derived from viral proteins. They have attracted intense interest from disciplines ranging from soft-matter engineering, catalysis, biomineralization and templating, to medical imaging, diagnostics, drug delivery, and – most relevant to this thesis – vaccinology. Enveloped viruses are over-represented in viral pathogens adversely effecting human health outcomes, and vaccines for many of these harmful viruses are either absent or in need of improvement. By virtue of their size, structure, repetitive geometries and therefore regular presentation of antigen, and their excellent safety profile, VLPs may provide solutions to the challenges of designing efficacious vaccines against enveloped viruses. Finding alternative adjuvants (compounds or molecules co-administered with vaccine formulations to elicit a heightened immune response) may further boost the applicability of VLP vaccines. Characterization of physical features, cellular interactions, and humoral and cellular immune responses generated by VLP vaccine candidates is vital for their commercialization. A detailed understanding of physical, mechanical, and structural characteristics of self-assembling nanoparticles can aid in the design and modification of novel nanostructures for a variety of applications. This overall goal of this thesis is to provide detailed morphological and biophysical characterization of VLP vaccine candidates from hepatitis C virus and from dengue virus, and to determine the three-dimensional structure of a self-assembling protein nanoparticle. In Chapter 2, hepatitis C VLPs were produced, and their topological and mechanical attributes were determined using Atomic Force Microscopy (AFM) techniques. Ordered packing of viral protein was observed, as was the structurally dynamic nature of enveloped VLPs. Glycosylation of the particles was investigated with a lectin binding array, and the VLPs were found to display similar glycosylation patterns to native hepatitis C virus. The cell attachment and entry behaviour of these particles was determined using fluorescence-assisted cell sorting techniques. Elasticity of the hepatitis C VLPs was determined for the first time, and values were found to be similar to those reported for liposomes. In Chapter 3, the hepatitis C VLPs were used in AFM force spectroscopy experiments to functionalize AFM probes and interrogate interactions between VLPs and liver cells grown either in monolayer or in organoid cell cultures. Differences in the cells generated in these different cell culture systems were interrogated by fluorescent labelling of key cell surface proteins (receptors) and cell structures, visualized by confocal laser scanning microscopy. Cells in organoid culture were found to resemble in vivo cell architectures more closely than did cells in monolayer culture. Binding interactions between VLPs and cells were only measurable by AFM in the organoid cell culture systems. This provides further evidence that organoid cell culture systems should be adopted for in vitro experiments, to provide more relevant results. Chapter 4 details development of a novel self-adjuvanted dengue VLP vaccine, again providing detailed characterization by AFM methodologies. A unique cloning strategy was employed, leading to properly matured and therefore appropriately immunogenic VLPs. Epitope presentation on these VLPs was assessed using binding assays with monoclonal antibodies directed to conformationally dependent epitopes on native dengue virus, and the VLPs were found to faithfully reproduce conformationally dependent dengue epitopes. Immunological responses to these nanoparticles were examined in vitro, and in vivo in a mouse model. VLPs were found to be highly immunogenic alone, with the inclusion of the adjuvant seen to significantly increase both cellular and humoral immune responses. The three-dimensional structure of a self-assembling protein nanoparticle, hemocyanin from the Australian freshwater crustacean Cherax destructor, was determined by CryoEM in Chapter 5, with supporting AFM studies. Hemocyanins are large respiratory proteins which form multimeric supramolecular complexes in a manner analogous to virus self-assembly. Hemocyanins from various organisms have been investigated for a number of biomedical and biotechnological applications. Hemocyanin from C. destructor was found to form a hexamer of monomer protein subunits, the dimensions of which could be approximated as a short cylinder of 10 nm height and 15 nm diameter. In-depth analysis was conducted of interface interactions between protein subunits, shedding light on the mechanism of self-assembly. In summary, the findings presented in this thesis provide baseline data and methodologies for measuring physical and mechanical qualities of VLP vaccine formulations to allow assessment of genetic modifications, or storage and delivery conditions, and should advance uptake of these important vaccine platforms. Cell cultures suitable for conducting in vitro investigations of viruses are identified, enabling cost- and time-saving relevant data to be obtained before moving to in vivo models. Amino acid level understanding of interactions involved in self-assembly in nanoparticles will support efforts toward design and modification of novel molecules for various applications.