Adherence to daily dosing regimens has emerged as a critical factor driving the clinical success of HIV-1 pre- exposure prophylaxis (PrEP) with antiretroviral (ARV) drugs in susceptible, uninfected individuals. This challenge can be mitigated with sustained release or long-acting ARV formulations that reduce dosing frequency, ideally to intervals of once per month or longer. Several ARV drugs are undergoing clinical evaluation as injectable sustained release formulations, but suffer from a number of drawbacks: a high initial concentration burst; the particles cannot be removed following injection should there be an adverse reaction; the approach requires specific ARV physiochemical characteristics, dramatically limiting the range of candidate drugs. Four recent large-scale clinical trials have shown that PrEP using preparations of the nucleoside reverse transcriptase inhibitor (NRTI) tenofovir (TFV) can prevent HIV-1 infection in a significant proportion of individuals. A long-acting TFV formulation for systemic dosing would add a much-needed NRTI to the portfolio of sustained release PrEP options. The low bioavailability of TFV to target immune cells supporting HIV-1 replication and the drug's high aqueous solubility make developing a long-acting formulation extremely challenging. Our proposal overcomes these hurdles by using the highly potent prodrug TFV alafenamide (TAF) delivered from a novel, patented, subcutaneous implant technology that provides linear release kinetics with no initial burst effect. In preliminary studis, we have developed a prototype TAF implant and evaluated its pharmacokinetics (PKs) in beagle dogs over 40 days. The implant maintained steady-state concentrations of TFV diphosphate (TFV-DP), the drug's active metabolite, in peripheral blood mononuclear cells that were thirty times higher than required for putative HIV-1 prophylaxis. The proposed efforts build on these important results and will test the central hypothesis that a one-year TAF implant with practical physical dimensions can safely prevent sexual HIV-1 infection. In Aim 1, we will design TAF implants for dose-ranging studies in mice, dogs, and macaques. We will work with a CMO to transfer the fabrication technology to build the capacity for manufacturing the implants under cGMP at the end of the project's five-year term. In Aim 2, we will evaluate the PKs and safety of the prototype implants in mouse, dog, and macaque models. Matrix-assisted laser desorption imaging mass spectrometry will be used to determine the 3D distribution of TFV and TFV-DP in vaginal and rectal tissues. Together, these foundational scientific studies will allow us to develo human PK simulation models that enable prediction of in vivo release rates from corresponding in vitro data. In Aim 3, HIV-1 prevention efficacy studies will be carried out in humanized mice and macaques, allowing the PK-pharmacodynamic relationships to be investigated in exploratory models. The above activities will be milestone-driven, culminating with submission of an Investigational New Drug (IND) application to the US FDA, allowing the technology to rapidly advance into clinical trials following the project's successful completion.