Formulation and development of recombinant protein vaccines

Recombinant protein vaccines are fast becoming the focus of the vaccine industry due to their increased safety. Here we examine the development of several recombinant protein vaccines and the challenges involved. Once an antigen is identified, and a process to recombinantly produce the protein established, the first step is to perform a biophysical characterization of the macromolecule. The proteins were stressed with respect to variables such as temperature and pH and monitored for perturbations in physical structure indicating potential sources of instability. Next, we examined aspects of formulation such as excipient screening and adjuvant adsorption which may enhance the antigen immunogenicity and stability. The third step involved evaluating the working formulation in an animal model to establish dose dependency and the effect of the adjuvant. Finally, accelerated and real-time stability studies are being completed, and the formulations adjusted accordingly.;We begin with three analogous mutant proteins from the pathogenic bacteria Shigella flexneri, Burkholderia pseudomallei and Salmonella typhimurium. All three species rely on a type III secretion system (TTSS), commonly referred to as a supramolecular injectisome, for virulence. This macromolecular complex is composed of 25 or more proteins which form basal and extracellular domains, and shares gross architectural similarities with bacterial flagella. The extracellular component or 'needle complex', previously identified as a potential vaccine target, is primarily composed of a single monomeric subunit organized in a helical array to form a hollow pore which protrudes from the bacterial membrane. Results of the biophysical characterization studies indicate that the secondary structure is largely alpha-helical in all three proteins, and surprisingly thermally labile with transition midpoints in the range of 35-50 °C over the pH range of 3-8. Second derivative UV absorbance spectroscopy data indicates some disruption of the protein's tertiary structure occurs at temperatures in the range of 29-46 °C. It appears, that at physiological temperatures, all three proteins experience intermediate nonnative molten globule like states in which they display significant secondary structure in the absence of extensive tertiary interactions. These antigens are found to be thermally stabilized by the presence of carbohydrates and polyols, and additionally all adsorb readily to aluminum hydroxide apparently through hydrogen bonds and/or Van der Waals forces. We have found that the interaction of the proteins with the adjuvant changes with time resulting in varying extents of irreversible binding. Peptide maps of desorbed protein, however, suggest that chemical changes are not responsible for this irreversibility. We also demonstrate the ability of MxiHDelta5 and PrgI Delta5 to elicit strong humoral immune responses in a murine model when administered as three intramuscular injections. When administered as monomers, the needle components exhibited strong dose dependent behavior, while the polymerized version (shown only for MxiH) was exceptionally immunogenic at low doses.;The second system described here is a recombinant ricin vaccine. There is an urgent need for the development of protective countermeasures against the use of ricin toxin as a bioterrorism agent due to its ease of access and distribution as well as its low lethal dose. We describe here the characterization of the stability of RiVaxRTM, an aluminum salt adsorbed recombinant ribotoxin A-chain double mutant, and optimization of adjuvant-antigen interactions which has allowed us to produce a stable vaccine that displays strong immunogenicity in mice. We used front face fluorescence as a physical measure of protein stability and monitored the adsorbed product upon storage at various temperatures. Indications of protein unfolding were observed and in the most extreme cases correlated with a decrease in immunogenicity of the vaccine. By adding phosphate anion, we are able to prevent the conformational changes, and maintain immunogenicity of the vaccine during long term storage.