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Title: Structural analysis of recombinant Caf1 and PA in alhydrogel-adsorbed and free forms
Author: Soliakov, Andrei
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2010
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In many cases conventional vaccines, based on live attenuated micro-organisms or killed whole cell preparations, are now being superseded by a new generation of subunit vaccines, which contain one or more highly purified recombinant protein antigens that offer a substantial improvement in safety, protection and manufacturing costs. Since purified proteins are often poor immunogens they need to be formulated with an adjuvant, a substance that non-specifically augments the immune response, to form the vaccine. Despite intensive research into novel adjuvants, still the most common clinical adjuvants are aluminium based hydrated gels. These aluminium gels readily adsorb protein antigens from aqueous solutions, forming insoluble protein-adjuvant micro-complexes. Yet despite their extensive use in vaccine formulations, very little is known of how these adjuvants affect the structure and stability of proteins. Thus there is urgent need for new biophysical analysis methods to characterise this final drug product. The work presented in this thesis aims to address this issue. Two model subunit vaccines were evaluated; namely recombinant capsular antigen fraction 1 (rCaf1) and recombinant protective antigen (rPA) used in new recombinant subunit vaccines against plague and anthrax, respectively. The interaction of rPA and rCaf1 with the Alhydrogel adjuvant was investigated by limited proteolysis, fluoresce nce, chemical modification, differential scanning and isothermal titration micro-calorimetry, electron microscopy and image processing techniques. The results reveal that protein structure is largely retained while cooperativity of folding is reduced. These small perturbations in protein structure can be modulated by addition of phosphate, in agreement with previous data from potency studies. The methods used here reveal new insights into how proteins interact with adjuvants and extend the capability for the characterisation of subunit vaccines. Furthermore, a combination of negative stain transmission electron microscopy and linear dichroism determined that the quaternary structure of Caf1 is a long flexible polymer up to 1.5 um long. It was found that donor strand exchange at the cohesive ends of shorter Caf1 polymers produces circular molecules. Site directed mutagenesis was used to study the unusual circular dichroism spectrum of Caf1. The contribution of aromatic residues in the far- and near ultra-violet spectra was assessed by circular dichroism and the protein fold was examined by Raman optical activity. This revealed unexpected contributions of aromatic residues to both the far- and near-ultraviolet circular dichroism spectra.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available