SUMMARY, Project 4 Alphaviruses regularly threaten global health, including long-established emerging viruses like Venezuelan equine encephalitis virus (VEEV, the most important of the encephalitic alphaviruses) and chikungunya virus (CHIKV, the most important arthritogenic alphaviruses) that unexpectedly emerged and spread globally. Moreover, other alphaviruses also threaten to emerge into urban epidemic cycles and spread extensively. To control these outbreaks and prepare for future alphavirus pandemics, we developed a new vaccine platform that uses the insect-specific, vertebrate cell replication-incompetent alphavirus, Eilat virus (EILV), as a genetic backbone for chimeras containing the structural polyprotein of target alphaviruses. CHIKV, VEEV, and eastern equine encephalitis virus chimeras are thermostable and provide rapid, durable, single-dose protection, advancing our goal of bridging the tradeoff between safety and rapid protection. A single dose of EILV- CHIKV induced neutralizing antibodies and conferred protection against CHIKV challenge one year after administration in non-human primates (NHPs). Here, in Project 4, we will extend these promising findings to demonstrate that the EILV platform can be applied rapidly and predictably to diverse alphaviruses to quickly generate safe and effective vaccines for future outbreak control. First, we will optimize the production protocols, doses, and routes of vaccination for EILV-VEEV. We will perform detailed assessments of EILV-VEEV immunogenicity in mice by measuring serum IgG binding and neutralization titers, and specific T cell responses. We will also test the ability of EILV-based vaccines to prime germinal center (GC) B cell, T follicular helper cell (Tfh), long-lived plasma cell (LLPCs), and memory B cell (MBC) responses. To identify early predictors of immune durability, we will measure longitudinal serum neutralizing antibody titers as well as the induction, magnitude, and specificity of antigen-specific MBC and GC B cells, recovered via fine needle aspirate of lymph nodes at early and late time points post-vaccination. To demonstrate an efficient, rapid workflow leading to a completely new, safe and immunogenic EILV-based vaccine for future outbreak control, we will develop a new chimeric EILV-based vaccine for western equine encephalitis virus vaccine, which is in a different antigenic alphavirus complex from CHIKV and VEEV. Finally, we will evaluate optimized EILV-based vaccines as a platform for cross-protection against diverse alphaviruses by developing blended formulations and prime- boost regimens. Lead multivalent (blended) candidates identified with mouse experiments with pan-alphavirus potential will be advanced to NHP efficacy testing with Core D, and a lead nanoparticle or mRNA vaccine candidate developed in Project 3 will be tested with the lead EILV-based vaccines in heterologous prime-boost regimens. By the end of this project, a rapid, safe, refrigerator-stable, and highly efficacious, single-dose vaccine platform will generate vaccines poised for final preclinical testing and a modular approach to address future alphavirus threats.