E-Book Overview
State-of-the-art review articles by leading experts summarize how to develop and employ the highly promising new DNA vaccines, what clinical results can be expected from their use, and what is known about how they work. Key topics range from vaccine design and construction to preparation and delivery methods, including the use of classical adjuvants, "genetic adjuvants," and the immunostimulatory properties of DNA and selected oligonucleotide sequences. Several contributors provide strategic ideas on antigen engineering and describe the novel applications of DNA vaccine methodology that have recently emerged. Cutting-edge and comprehensive, DNA Vaccines: Methods and Protocols provides a snapshot of the methods and thinking from which the vaccines of tomorrow will evolve.
E-Book Content
1 Purification of Supercoiled Plasmid Anthony P. Green 1. Introduction Current technologies for the purification of supercoiled plasmids are limited. The use of cesium chloride gradients in the presence of ethidium bromide is time consuming, labor intensive, requires the use of known mutagens and is not conducive to large scale. As a result, first-generation high-performance liquid chromatography (HPLC) methods based on anion-exchange and size exclusion have been developed but are difficult to accommodate production at large scale and still result in compromised purity (1,2). The success of DNA vaccines in animal models and the initiation of human trials (3,4) has led to a need to increase the level of supercoiled plasmid purity as well as the methodology utilized to produce these plasmids at large scale. Several parameters of the purification process need to be addressed: • The ability to prepare supercoiled plasmid at purity levels acceptable for clinical material. • The ability to prepare clinical grade supercoiled plasmid that will be scalable in order to produce gram quantities of produc