PROJECT SUMMARY The objective of this project is to develop DNA nanoparticles with efficient delivery to the local draining lymph nodes (dLN) via subcutaneous (s.c.) administration to promote dendritic cell (DC) transfection, antigen presentation, and T cell activation in the lymph nodes, and to elicit robust antibody titers and immunological memory against Zika virus. As the Zika virus (ZIKV) epidemic in Brazil spread around south and north Americas, Europe, and Asia, teams around the world have been racing to develop ZIKV vaccines. DNA vaccines offer many advantages in terms of ease of production, excellent stability with long shelf life, multivalent capability, and fast development cycle. The first generation of vaccine candidates is built with the pre-membrane and envelope sequences from a Brazilian ZIKV strain as the dominant immunogen. They have been tested as naked plasmid DNA vaccines administered by intramuscular (i.m.) injection, and generated effective neutralizing antibodies in mice and nonhuman primates. However, naked DNA vaccine inherently has low efficiency to generate Th2 response and immune memory, and requires higher DNA dose due to the low abundance of antigen presenting cells (APCs) at the injection site. This study will directly address these challenges by combining two novel approaches to engineer a more potent ZIKV DNA vaccine: (1) a chimeric DNA construct encoding ZIKA envelope protein sequences and lysosomal-associated membrane protein-1 (LAMP), which can direct the expressed antigen to MHC Class II-rich compartment, thus skewing ZIKV antigen presentation towards a strongly Th2-biased response and generation of immune memory; and (2) flash nanocomplexation (FNC)-produced small (~40 nm) ZIKV/LAMP DNA nanoparticles to enable drainage to the dLNs following s.c. injection, thus enhancing the immune response and allowing for substantial reduction in vaccination dose. We will first engineer the DNA nanoparticles with different sizes and narrow distribution using the FNC method, and characterize their stability, surface properties, and ability to transfect DCs; then determine the effect of NP size on LN-draining efficiency and gene expression in DCs in different dLNs; and lastly demonstrate the robust immune responses elicited by LN-targeting NPs with LAMP/ZIKV DNA vaccine, and characterize the subtypes of anti-ZIKV response. This timely study will not only develop a more effective ZIKV vaccine and support further testing of the next generation of Zika vaccine, but also provide a potent DNA vaccine platform against other emerging pathogens.