Design and Purification of Subunit Vaccines for Prevention of Clostridium difficile Infection

Clostridium difficile is a gram-positive bacteriumresponsible for a large proportion of nosocomial infections in the developed world. C. difficile secretes toxinsA and B (TcdA and TcdB) and both toxinsact synergistically to induce a spectrum of pathological responses in infected individuals ranging from pseudomembranous colitis to C. difficile-associated diarrhea. ToxinsA and B have been actively investigated as components of prophylactic vaccine as well as targets for therapeutic intervention with antibodies. Expression of such toxinsby recombinant technology is often difficult and may require special handling and adherence to strict safety regulations during the manufacturing process due to the inherent toxicity of the proteins. Both toxinsare large proteins (308 kDa and 270 kDa, respectively) and contain distinct domains mediating cell attachment, cellular translocation, and enzymatic (glucosidase) activity. Here we describe methods to produce fragments of ToxinB for their subsequent evaluation as components of experimental C. difficile vaccines. Methods presented include selection of fragments encompassing distinct functional regions of ToxinB, purification methods to yield high quality proteins, and analytical evaluation techniques. The approach presented focuses on ToxinB but could be applied to the other component, ToxinA, and/or to any difficult to express or toxic protein.