Recombinant poxviruses: versatile tools for immunological assays

The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus (VACV), a poxvirus closely related to Cowpox, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviruses readily infect a variety of cell types, and they drive rapid and high levels of antigen expression. Additionally, they can be utilized in a variety of assays to study both MHC class I-restricted and MHC class II-restricted antigen processing and presentation. Ultimately, the numerous advantages of poxvirus recombinants have made the Vaccinia expression system a mainstay in the study of processing and presentation over the past two decades. In an attempt to address one shortcoming of VACV while simultaneously retaining the benefits inherent to poxviruses, our laboratory has begun to engineer recombinant Ectromelia viruses. Ectromelia virus, or mousepox, is a natural pathogen of murine cells and performing experiments in the context of a natural host-pathogen relationship may elucidate unknown factors that influence epitope generation and host response. This chapter describes several recombinant poxvirus system protocols used to study both MHC class I and class II antigen processing and presentation, as well as provides insight and troubleshooting techniques to improve the reproducibility and fidelity of these experiments.