Recombinant Vaccinia Virus with Reduced Virulence

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Vaccinia Virus (VV)-Wyeth base vaccines for the prevention of small pox have a margin of safety over other dependable vaccine strains. In contrast, specifically attenuated VV do not offer robust protection. The objectives of this proposal are use contemporary molecular genetics to specifically mutate the VV-Wyeth strain so as to: (i) Lower virulence by specifically deleting immune modulatory and anti-inflammatory functions, while maintaining host range, replication, and immunogenicity as measured by replication in primary human fibroblasts and keratinocytes, MRC5. Vero, RK13. BHK and NIH3t3 cells and by replication and immune potency in murine models of IP, intranasal, or dermal scarification VV infection. The Kieff lab has made recombinant VV for expression of Epstein-Barr Virus genes in the past and has collaborated with the Welsh lab in characterizing the effect of v-ILl0 on VV virus infection and immune responses. The Welsh lab has studied mouse responses to VV infection for over 20 years, discovering the activation and antiviral role of NK cells and NK cell subsets, the activation and antiviral role of 7_ T cells, heterologous immune response, whereby memory a[_ T cells specific to other viruses alter VV pathogenesis, and (in collaboration with Dr. Liisa Selin) defined a class I Kb - restricted, immunodominant, VV epitope. The kinetics of VV synthesis, dissemination, immune responses, and morbidity following systemic and mucosal infection of C57BL/6 mice are already well defined. Genetically manipulated C57BL/6 mice with altered immune system components, such as antiviral cytokines and lymphocyte subsets, will assist in evaluating how an immune- compromised hosl may respond to a more attenuated, but still immunogenic vaccine. Mice expressing human HLA-A2 are also available and will be used to assess the significance of human T cell epitopes. We aim for novel insights into VV virulence and expect to define mutations that will be useful for human vaccines. (ii) Increase VMV immune potency by including potential VMV T cell epitopes that are missing from VV. These experiments will be done in collaboration with Professors Welsh and Reinherz and will initially focus on A2 epitopes because of the utility of the human HLA-A2 mice. And (iii) Increase knowledge of VV virulence and pathogenesis, by investigation of the molecular genetic and immunological effects of specific VV open reading frames that are prime candidates for immune modulation based virulence.