CONSENSUS GII.4 VIRUS LIKE PARTICLES; A VACCINE CANDIDATE BIOPHYSICAL CHARACTERIZATION, STABILIZATION, AND ADJUVANTS BINDING STUDIES

Abstract

The World Health Organization (WHO) and the United Nations Children's Fund (UNICEF) estimate globally that 1.9 million children under the age of five years old die annually as consequence of diarrhea. Increasing need for hospitalization, which has negative effects on health care sectors, in addition to mortality incidences mark diarrheal related diseases a growing burden on both health care sectors and the global economy. Introducing novel measures to control and avert the spread of diarrheal disease are of paramount importance. Diarrhea is the main symptom in Acute gastroenteritis (AGE) and according to the United States' National Outbreak Reporting System (NORS), viral agents are the dominant precursors of epidemic AGE outbreaks. More than 90% of humans' acute non-bacterial gastroenteritis breakouts worldwide is caused by Noroviruses (NoVs). NoVs of the Caliciviridae family are none enveloped, positive sense, single stranded RNA virus, noncultivable in cell culture, containing three open reading frames (ORF). Expressing NoVs ORF2 in a baculovirus expression system produces empty virus-like particles (VLPs). These VLPs are very similar to the native virus in terms of morphology and antigenicity, yet lacking the genetic material essential for infectivity, making the VLPs a superb candidate for vaccine development. By comparing the capsid sequence of three different NoVs GII.4 strains, consensus GII.4 VLPs were produced as a potential vaccine with the goal of providing a broader protection against AGE. The main objective of this study is to understand the structural behavior of NoVs Consensus GII.4 VLPs, which is to be used as a vaccine. A complement of biophysical techniques has been employed to characterize the physical stability of Noroviruses Consensus GII.4 virus-like particles (VLPs) as a function of temperature and pH. The VLPs' physical stability are characterized by different spectroscopic techniques and the resulting data are used to construct empirical phase diagrams (EPDs) projecting the entire data set in the form of a colored image. These EPDs are used in the development of excipient screening assays to identify potential stabilizers of the VLPs in solution. The identified stabilizers are then subjected to further screening using fluorescence analysis to determine their optimal concentrations and use in combination. The generated data are used to construct binding isotherms for Consensus GII.4 VLP and aluminum salt adjuvants (Alhydrogel® and Adjuphos®). Binding isotherms were also generated for Norwalk VLP (a previously studied vaccine candidate) and aluminum salt adjuvants. Front Face Fluorescence Spectroscopy is used to evaluate the structural changes associated with Consensus GII.4 and Norwalk VLPs when bound to aluminum salt adjuvants.