We propose a new category of viral vaccines consisting of replication competent viruses with pseudolethal mutations that render them incapable to form the infectious progeny. We suggest, that since such viruses will retain the capabilty for intracellular replication, they can induce immune responses comparable to that resulted from infection. This class of vaccines cannot be prepared and used as "regular" infectious vaccines. However, if made in the infectious DNA form, such vaccines could combine the efficiency of infectious viruses in induction of protective immune responses, the safety of non-infectious vaccine preparations, and the low cost of DNA vaccine production and maintenance. The following objectives will be addressed in the proposed study. 1) We will design flavivirus infectious DNA constructs capable of initiating productive infection upon transfection of plasmid DNA into susceptible cells and inoculation into mice. We have developed a revolutionary approach affording design of remarkably stable infectious DNA constructs of flaviruses. We will prepare infectious DNA of West Nile (WN) virus, which recently emerged in United States and vaccine for which is not available, and of the 17D vaccine strain of yellow fever (YF17D) virus, which is very attractive as a vaccine vector. 2) We will introduce mutations into WN and YF17D infectious DNA constructs that will render these viruses incapable of forming infectious progeny. We will introduce deletions in the capsid gene that will abolish virion assembly, and/or modify the site at C-prM junction cleaved by the viral protease prior to or during virion assembly. Such modifications will not affect earlier processes of RNA replication, synthesis and processing of viral proteins, thus leaving the intracellular phase of viral replication largely intact. We will characterize replication and infectious properties of such mutants. 3) Using DNA immunization methodology, we will evaluate the immunogenic potential of mutated WN and YF17D viruses that will be capable of only a single round ol intracellular replication, and compare with that of a "classic" non-replicating DNA vaccine and of an infectious virus. Using ELISA, Western blot, and FAGS, we will characterize humoral, GIL and Th1/Th2 profiles of the resulting immune responses. Finally, we will determine their protective potential in virus challenge experiments. The WN infectious clone will facilitate development of a WN vaccine, and one candidate will be evaluated in the course of this study.