Title: Engineering antiviral innate immunity for safe and effective microbicides HIV infections afflict millions of people and cause tremendous health and economic burdens. One of the major risk factors for HIV-1 transmission is the pre-existing infections caused by sexually transmitted agents such as herpes simplex virus type 2 (HSV-2). Therefore, a rational prevention strategy to halt HIV spread is to target HSV-2 infection and control its spread. In the absence of vaccines against HSV-2, a more practical and effective intervention for HSV-2 is the utilization of microbicides. A promising microbicidal approach is to potentiate antiviral innate immunity effective against a broad range of viruses at the site of viral encounters. The toll-like receptor (TLR)-based innate immunity have been shown to be crucial in initiating a cascade of antiviral activities mediated by type I interferons (IFNs). Both TLR3 and TLR9 agonists, polyinosinic:polycytidylic acid (poly IC) and CpG oligonucleotides (ODNs), are effective in protection against HSV-2 infections. However, undesirable inflammatory responses and autoimmunity accompanying the non-specific stimulation of TLRs are of major concern, which could severely limit the use of TLR agonists as microbicides. Thus, the key to developing TLR agonists as microbicides is to target them to relevant cell types at the potential sites of viral exposure and to elicit IFN responses in a regulated fashion. We propose to develop localized, controlled-release, and cell-targeted delivery systems to regulate the stimulation of TLR-based innate antiviral immunity. In the R21 Phase, three aims will be accomplished: Aim 1: to design and characterize cell-targeted delivery systems based on poly (lactide-co-glycolide) (PLGA) nanoparticles to specifically and locally target pDCs and epithelial cells with TLR agonists; Aim 2: to evaluate the effectiveness against genital HSV-2 infections by locally and selectively targeting CpG ODNs and/or poly ICs to pDCs and epithelial cells with cell-targeted nanoparticles; Aim 3: to evaluate toxicityby locally and selectively targeting CpG ODNs and/or poly ICs to pDCs and epithelial cells with cell-targeted nanoparticles. Built upon the results from the R21 phase, in the R33 phase, we will accomplish: Aim 4: to design and characterize delivery systems for sustained release of TLR agonists; Aim 5: to evaluate the effectiveness against genital HSV-2 infection and toxicity by localized, sustained-release and cell-targeted nanoparticles loaded with CpG ODNs and/or poly IC; Aim 6: to evaluate the adaptive immunity against genital HSV-2 infection mediated by localized, sustained-release and cell targeted nanoparticles loaded with CpG ODNs and/or poly IC. This application will enable the translation of TLR-based antiviral innate immunity to effective and safe microbicides.