The Phase I studies established the feasibility of using molecular approaches to derive from the NYCBH vaccine strain, stable attenuated mutants of unaltered immune efficacy. In this Phase II proposal selected mutants generated in the virulent NYCBH-IH isolate will be studied to determine their virulence parameters and immune protective capacity. An exhaustive study of virulence parameters in normal and immunocompromised mice will include, intracranial and intranasal LD50s, growth and dissemination from the site of inoculation, and the extent of pock formation on scarified skin. The immune protective capacity of mutants will be assessed by their ability to confer protection against a foreign pathogen by the expression of a single neutralizing antigen. The studies described above were performed for experimental convenience in a high virulence laboratory isolate of NYCBH. This information will be used to create specific mutations in a low virulence plaque purified isolate form the Wyeth vaccine seed stock (NYCBH-W) produced for the NIH under good manufacturing practice. The NYCBH-W mutants will be assessed for virulence parameters, and ability to confer protection against a foreign pathogen by the expression of a single neutralizing antigen. This data will be used to design first generation vaccine candidate(s) containing single mutations. Subsequently, combinations of mutations will be used to conform second generation vaccine candidate(s).