DNA-only vaccination would significantly enhance the rapid deployment utility of DNA vaccines for pandemic application (development times for DNA vaccines are significantly shorter than those for protein or viral vector systems). The overall goal of this project is to develop a rapid deployment DNA vaccine platform protective against pandemic influenza H5N1. The hypothesis is that influenza DNA vaccine potency can be increased through activation of cytoplasmic double stranded RNA (dsRNA) signaling pathways by vector encoded immunostimulatory RNA. This will be accomplished through the use of various immunostimulatory "RNA elements" (RNAe). This Phase I proof of concept study will investigate the potential benefit of inclusion of vector-mediated RNAe on immune responses to an vector encoded target antigen. Influenza Hemagglutinin (HA) will be used as an example antigen requiring antibody responses for protection. Novel HA DNA vaccine plasmid vectors that produce immunostimulatory dsRNA will be created and screened for immune response in vivo after delivery by electroporation-enhanced intramuscular injection. The optimal backbone that improves neutralizing antibody response to HA will be selected. Then, building upon this RNAe vector, 'inhibiting-the-inhibitor' DNA vaccines will be created and tested in a subordinate study. The secondary hypothesis is that immune responses induced by the dsRNA activating DNA vaccines can be further improved by preventing target cell downregulation of immune responses mediated by vector activation of innate immunity. The vectors defined above will be further modified to incorporate RNAe which knockdown expression of various attenuators of these cytoplasmic dsRNA signaling pathways. Antibody responses to HA induced by the 'inhibiting-the- inhibitor' DNA vaccines will then be determined in an animal model. In Phase II the resulting optimal vector backbone (i.e., specific immunostimulatory RNAe alone, or potentially in combination with 'inhibiting the inhibitor' shRNA as determined herein) will be developed as a novel, rapid deployment DNA-only vaccine platform for influenza H5N1. The rapid deployment DNA vaccine platform developed herein will have general utility with a variety of other emerging biological agents; NTC will license the platform for such applications. This proposal is aimed at developing novel influenza vaccines to replace egg based vaccine production technologies and as such is responsive to NIAID's high priority influenza research areas, as outlined in NOT-AI-06-011
