ABSTRACT: The fully cured Berlin patient example instilled a lot of optimism in the HIV field and the current thrust is to forge ahead with developing novel HIV-1 eradication strategies. In this context, it is essential that reliable tests are developed that can verify significant reductins in inducible viral reservoirs and predict responses to curative strategies after antiretroviral therapy (ART) interruptions (ATI). Other than ATI, viral out growth assays (VOA) that screen for latently infected cells by co-culturing methods have been critical to quantifying inducible or replication competent residual virus that may persist after a curative strategy. However, in some patients with prolonged undetectable plasma and cell-associated HIV-1 such as the two Boston patients who underwent allogeneic HSCT and the Mississippi child who had undergone early ART right after birth, traditional VOAs could not detect residual virus in peripheral CD4 T cells although virus rebounded after treatment interruption. Thus, it has become important that more sensitive VOAs that employ novel and innovative systems such as HIV susceptible humanized mice (hu-mice) need to be developed and validated. In a recent development, latent virus was successfully recovered from fully virus suppressed SIV infected macaques (as determined by all standard tests) undergoing intensive ART by adoptive transfer of their CD4 T cells to naive animals. This showed that ultralow levels of otherwise undetectable latently infected cells could be captured and induced using an in vivo system. Our current proposal is based on these recent findings and the hypothesis that an in vivo humanized mouse viral outgrowth assay (hmVOA) will be more sensitive than traditional in vitro VOAs at detecting low levels of persistent infectin in HIV patients. Our specific aims are to: 1. Develop and optimize an in vivo humanized-mouse viral outgrowth assay (hmVOA) for latent HIV using patient derived resting CD4 T cells, 2. Compare hmVOA with traditional VOA using patient derived resting CD4 T cells and determine if hmVOA is more sensitive, 3. Determine whether hmVOA can detect persistent HIV infection in patients in which virus is undetectable by any other method, including VOA, 4. Evaluate if hmVOA can more readily detect virus-latent cells from tissue reservoirs/ sanctuaries of long-term fully suppressed patients' compared to standard VOA, 5. Determine if sensitivity of hmVOA can be further improved by in vivo treatment of cell infused mice with HIV latency-reversing agents such as romidepsin and panobinostat.