This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Alphaviruses including Venezuelan (VEEV), eastern (EEEV) and western equine encephalitis viruses (WEEV) are highly developed agents of biological warfare and terrorism (BWT) and important, naturally emerging zoonotic viruses. Current biodefense against these viruses is inadequate, and widespread morbidity and mortality could be inflicted upon exposure of civilians or military personnel. Effective, licensed vaccines, critical first lines of defense and important tools for biodefense research, are badly needed. We have focused on live virus vaccines to take advantage of their high levels of antigen presentation, their ability to induce both humoral and cell-mediated immunity, and the faster and longer lived antibody levels characteristic of exposure to replicating viruses. In the proposed continuation of our alphavirus vaccine development project, we will refine and further validate the safety and efficacy of a set of vaccine candidates against VEEV, EEEV and WEEV. Additional attenuation strategies will be tested in the chimeric vaccine genomes to take advantage of our recent determination that the capsid proteins of some alphaviruses inhibit host cell transcription, an important mechanism of virulence. As a further safety measure, a novel genetic strategy will be developed to completely debilitate the ability of the chimeric vaccine viruses to replicate in mosquito cells, both in vitro and in vivo. Finally, to further evaluate the potential of these chimeras for vaccination of humans, Cynomolgus macaques will be immunized with 2 different Sindbis/Eastern equine encephalitis vaccine candidates in a preliminary assessment of safety, immunogenicity and attenuation. In addition to protection of civilian and military populations from an anticipated alphavirus BWT event, and protection of laboratory personnel doing critical BWT and public health research, the methods we will develop can be exploited to rapidly and efficiently develop new vaccines against newly recognized, emerging alphaviruses or an engineered alphavirus weapon. They will also be useful for protecting populations at risk of natural exposure to these zoonotic agents in many parts in the New World. Thus far, we have completed an efficacy study using the candidate EEEv vaccines using cynomolgus macaques (Macaca fascicularis). Animals were primed with a Sindbis virus vectored North American (SIND-NAEEEvv) or South American (SIND-SAEEEvv) variant vaccine or sham immunized with saline. Animals were bled and antibody production (alpha-EEEv IgG) was determined. The animals were challenged with a North American origin EEE virus (FL-9393) by aerosol at a target lethal dose (1.0E+07 PFU inhaled). Animals receiving SIND-NAEEEvv variant were mostly protected (5/6 survival; 83%) whereas animals vaccinated with the South American variant (SIND-SAEEEvv) or sham-vaccinated (saline) died as a result of exposure (1/6; 16% and 0/4; 0% survival respectively). Telemetric monitoring of the animals post challenge showed the animals successfully vaccinated experienced minimal to no disruption in normal diurnal variation in core temperature or abnormalities in heart rate and ECG. The affected animals, alternatively, experienced significant changes (hyperthermia) in core temperature and disruption in ECG signal. Histopathological analysis of tissues, specifically the brain, indicated viral invasion and significant encephalitis. We are repeating this experiment in cynomolgus macaques to confirm the efficacy of SIND-EEEvv in this model.