Thomas R. Foubert

Conformational Stability and Disassembly of Norwalk Virus-like Particles EFFECT OF pH AND TEMPERATURE

Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus-like particles (VLPs) with a well defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants of the parent virion are preserved. They also constitute very simple models to study the mechanisms of assembly and disassembly of viral capsids. This work examines the inherent stability of NV-VLPs over a range of pH and temperature values and provides detailed insight into structural perturbations that accompany disassembly. The NV-VLP structure was monitored using a variety of biophysical techniques including intrinsic and extrinsic fluorescence, high resolution second-derivative UV absorption spectroscopy, circular dichroism (CD), dynamic light scattering, differential scanning calorimetry, and direct observation employing transmission electron microscopy. The data demonstrate that NV-VLPs are highly stable over a pH range of 3-7 and up to 55 °C. At pH 8, however, reversible capsid dissociation was correlated with increased solvent exposure of tyrosine residues and subtle changes in secondary structure. Above 60 °C NV-VLPs undergo distinct phase transitions arising from secondary-, tertiary-, and quaternary-level protein structural perturbations. By combining the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for NV-VLP was constructed. This strategy of visualization provides a comprehensive description of the physical stability of NV-VLP over a broad range of pH and temperature. Complementary, differential scanning calorimetric analyses suggest that the two domains of VP1 unfold independently in a pH-dependent manner.

Research ArticlesPhysical Stabilization of Norwalk Virus‐Like Particles*

Virus-like particles (VLPs) used as vaccine antigens often elicit strong immune responses due to their intrinsic repetitive, high-density display of epitopes, and the fact that the mammalian immune system is highly attuned to recognizing particles in the size range of viruses (20-150 nm). To retain these immunogenic qualities, vaccines that utilize virus-like particle (VLP) antigens should be formulated to stabilize both native conformational epitopes and the overall particulate nature of the VLP. This work describes a systematic approach for identifying potential stabilizers for formulation of Norwalk VLPs (NV-VLPs) in aqueous suspension. A number of excipients were screened for their ability to inhibit aggregation of NV-VLPs under conditions known to induce aggregation. Those compounds shown to inhibit aggregation were further evaluated under conditions of thermal stress and the NV-VLP structure was monitored using biophysical techniques such as CD, ANS fluorescence, and DSC to provide insight into the mechanisms by which stability was conferred. Increased thermal stability in the presence of chitosan glutamate, sucrose, and trehalose was correlated with stabilization of secondary and tertiary structural elements of NV-VLPs. These excipients may be useful for formulation of a stable NV-VLP vaccine.