PROJECT SUMMARY Antiretroviral therapy fails to cure HIV-1 infection due to a pool of latently infected cells that resume viral repli- cation upon treatment interruption. This has sparked the development of therapeutic approaches toward a cure for HIV-1 infection. Those entailing immune responses against viral epitopes require previous viral reactivation with latency reversing agents, which do not efficiently induce the expression of viral antigens. In recent years, techniques have been developed to identify epitopes naturally processed and presented by class I and class II HLA molecules (HLA ligandome) to elicit both CD4+ T helper and CD8+ CTL specific effec- tor and memory responses. This strategy has been applied in the cancer field, thus allowing the design of ther- apeutic vaccination strategies seeking to elicit cell-based immunity against tumor-associated peptides. This novel approach is currently being evaluated in the clinical setting to treat human cancers, such as glioblasto- ma, multiple myeloma, acute myeloid leukemia, hepatocellular carcinoma, ovarian and renal cell cancer. A comprehensive analysis of epitopes uniquely expressed on HIV-1 latently infected cells has never been at- tempted. Nevertheless, this might provide the basis for the development of immune-based therapeutic ap- proaches aimed at killing cells harboring latent HIV-1 in the absence of viral reactivation. The main goal of this application is to define the HLA ?ligandome? (viral and host epitopes) of latently infected compared to uninfected cells. The identification of such unique subset of peptides could lead to devise therapeutic vaccination strategies to target and eliminate latently infected cells in the absence of viral reactivation. To achieve this goal, we propose two specific aims. Specific Aim 1 seeks to identify the unique ligandome expressed on the surface of latently infected cells in the context of MHC-I and II molecules. We will obtain MHC-I and II-associated peptides expressed on latently infected and uninfected cells generated with an in vitro model developed in our laboratory, and we will identify them by mass spectrometry. Peptides uniquely expressed on latently infected central memory CD4+ T cells will be screened against a large data- base of peptides expressed on the surface of normal, healthy human cells. This will define the viral and host ligandome that uniquely characterizes CD4+ T cells latently infected with HIV-1. Specific Aim 2 will evaluate immune responses to specific peptides identified in the ligandome of HIV-1-latently infected cells in cART-treated HIV-1 infected subjects. We will assess the frequency of T-cell responses in HIV-1 chronically infected patients against selected epitopes. Specific responses will be assessed via IFN? and IL-4 intracellular cytokine staining, IFN? and IL-4 ELISPOT assay, and tetramer binding assay. Results will provide relevant da- ta in order to determine the extent of responses to the identified epitopes in HIV-1 latently infected patients. The studies proposed in this application will be conducted in collaboration with Drs. Luigi Buonaguro (National Cancer Institute, Italy), Maureen Kane (University of Maryland School of Pharmacy), and Mathias Lichterfeld (BWH/MGH, Boston), each contributing a unique set of expertise needed for the completion of the project.