Vaccinia Virus (VV)-Wyeth base vaccines for the prevention of small pox have a margin of safety over otherdependable vaccine strains. In contrast, specifically attenuated VV do not offer robust protection. The objectives of thisproposal are use contemporary molecular genetics to specifically mutate the VV-Wyeth strain so as to: (i) Lowervirulence 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, ordermal scarification VV infection. The Kieff lab has made recombinant VV for expression of Epstein-Barr Virus genesin the past and has collaborated with the Welsh lab in characterizing the effect of v-ILl0 on VV virus infection andimmune responses. The Welsh lab has studied mouse responses to VV infection for over 20 years, discovering theactivation and antiviral role of NK cells and NK cell subsets, the activation and antiviral role of 7_ T cells, heterologousimmune response, whereby memory a[_ T cells specific to other viruses alter VV pathogenesis, and (in collaborationwith 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 arealready well defined. Genetically manipulated C57BL/6 mice with altered immune system components, such asantiviral cytokines and lymphocyte subsets, will assist in evaluating how an immune- compromised hosl may respondto a more attenuated, but still immunogenic vaccine. Mice expressing human HLA-A2 are also available and will beused to assess the significance of human T cell epitopes.
We aim for novel insights into VV virulence and expect todefine mutations that will be useful for human vaccines. (ii) Increase VMV immune potency by including potentialVMV T cell epitopes that are missing from VV. These experiments will be done in collaboration with Professors Welshand 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 immunologicaleffects of specific VV open reading frames that are prime candidates for immune modulation based virulence.
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