| dc.description.abstract | Subunit vaccines contain highly defined macromolecular components of a pathogen that are capable of eliciting protective immunity. They possess several advantages over other vaccine types (e.g. live attenuated and inactivated) such as improved safety profiles, highly defined nature, ease of production, and potential for lower cost of goods. One critical limitation of subunit vaccines, however, is their weak immunogenicity owing to their inability to replicate, monovalent structures, and the absence of other immunostimulatory components. Common approaches to enhance the immunogenicity of subunit vaccines include polyvalent antigen display strategies, the use of adjuvants, etc. The polyvalent antigen display strategy requires the use of a scaffold, which can be protein-based or some other materials. Chapter 2 focuses on the biophysical properties of a potential scaffold for polyvalent antigen display-Bacillus anthracis lumazine synthase (LS), an icosahedral homo-oligomeric protein. LS in PBS buffer showed a minor thermal transition around 50 ᵒC, and a major one at 95 ᵒC. The minor transition arose from the dissociation of the LS/phosphate complex, which formed in PBS buffer at room temperature. The major transition corresponded to the dissociation of LS oligomers, thermal unfolding, and aggregation. In chapter 3, I describe an attempt to develop ricin vaccine candidates in which LS was used as a scaffold to achieve polyvalent display of a linear neutralizing epitope (designated PB10) from ricin. PB10 was genetically inserted onto the C terminus of LS, and the fusion protein (designated LS_PB10) was expressed in an E.coli system. LS_PB10 self-assembled into spherical particles. Fusion of the PB10 peptide did not affect the structure and stability of LS. LS_PB10 showed tight binding to a mAb targeting the PB10 epitope. Immunization of LS_PB10 in mice elicited a moderate level of anti-ricin serum titers, which, however, failed to offer protection during a challenge study using a 10x lethal dose of ricin. Such an unsatisfactory end result may be attributable to 1) limited efficacies of using the PB10 epitope alone, 2) loss of secondary structure of PB10 on LS; 3) an epitope suppression effect induced by the highly immunogenic nature of the LS scaffold. Studying antigen/adjuvant compatibility is critical for the development of adjuvanted vaccine formulations. Chapter 4 discusses the utilization of biophysical tools to understand effects of two emulsion-based adjuvants (designated as ME.0 and SE) on the structure and thermal stability of alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection. Both adjuvants are oil-in-water (O/W) emulsions using squalene as the oil phase. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a blue shift in Trp fluorescence emission spectra of AT. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. The goal of protein formulation development is to identify optimal conditions for long-term storage. Certain commercial conditions (e.g., high protein concentration or turbid adjuvanted samples) impart additional challenges to biophysical characterization. Formulation screening studies for such conditions are usually performed using a simplified format in which the target protein is studied at a low concentration in a clear solution. The failure of study conditions to model the actual formulation environment may cause a loss of ability to identify the optimal conditions for target proteins in their final commercial formulations. In chapter 5, we utilized a steady-state/lifetime fluorescence-based high-throughput platform to develop a general workflow for direct formulation optimization under analytically challenging but commercially relevant conditions. A high-concentration monoclonal antibody and an Alhydrogel-adjuvanted antigen were investigated. A large discrepancy in screening results was observed for both proteins under these two different conditions (simplified versus commercially relevant). This study demonstrates the feasibility of using a steady-state/lifetime fluorescence plate reader for direct optimization of challenging formulation conditions and highlights the importance of performing formulation optimization under commercially relevant conditions. | |
