Poxviruses, and in particular vaccinia virus, have been utilized as systems for the expression of proteins in eukaryotic cells and as vectors for antigen delivery. Their ability to incorporate large amounts of DNA and wide host range leads to the expression and correct processing of a great variety of proteins in many cell lines. High level expression vectors have been constructed by expressing bacteriophage RNA polymerase genes in vaccinia. These vectors express high levels of any gene located behind the bacteriophage promoter. We have developed a conditionally lethal vaccinia virus expression vector which expresses the T7 bacteriophage RNA polymerase. Under the restrictive conditions of elevated temperature, the virus undergoes an abortive infection where greatly reduced amounts of the vaccinia late or post-replicative genes are synthesized. We are quantitating the absolute level of expression of foreign genes in this system and comparing its utility to presently available vaccinia T7 expression systems. Also, this vector provides a means for the analysis of potential trans-acting factors participating in vaccinia late processes such as resolution of DNA replicative intermediates. Poxvirus vectors are customarily constructed by introducing, the foreign DNA into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been used to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We have constructed and characterized a new set of direct ligation vectors. These vectors contain restriction sites to fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters at the beginning of the thymidine kinase gene in vaccinia to utilize drug selection in the isolation of recombinants. These viruses provide a set of universally applicable, direct ligation, poxvirus cloning vectors extending the utility of poxvirus vectors for construction and expression of complex libraries. Current efforts are concentrated on the application of this approach to the live attenuated virus modified virus Ankara (MVA) This lab is working on the development of new poxvirus based vectors. This includes the development of direct ligation vectors for efficient chimera construction and the design of attenuated viruses with host range defects to increase their safety, especially for immunocompromised hosts. One host range restricted attenuated virus under consideration as a vector is modified virus ankara (MVA). The laboratory has participated in research aimed at determining the viral gene products missing in MVA which lead to the altered host range phenotype.
