Highly active antiretroviral therapy (HAART) effectively reduces HIV-1 replication, but the early optimism for viral eradication through prolonged treatment with these drugs has not been met. This is largely due to the fact that HIV-1 can cause a latent infection of host cells that develops when infected, activated cells revert back to a quiescent state. These latently infected cells represent a transcriptionally silent reservoir for HIV-1 infection that is resistant to currently available antiretroviral drugs and HIV-1-specific immune responses. The prolonged half life of latently infected cells (approximately 44 months), combined with their ability to rapidly initiate virological rebound after discontinuation of HAART, are the main factors responsible for viral long-term persistence. These characteristics are in sharp contrast to lytically infected cells, which represent a highly activated, short-lived cell population with high viral production that can be effectively suppressed by antiretroviral drugs and HIV-1-specific CD8+ T cells. Understanding the molecular mechanisms that govern the maintenance, survival, turnover and HIV-1 gene expression in latently and lytically infected cells would open up novel perspectives for developing specific therapeutic options to target cells with persistent viral replication or latent HIV-1 infection, and therefore represent one of the highest priority topics in current HIV-1 research. The major technological obstacle to characterizing latently infected cells in sufficient detail is our current inability to identify and physically isolate these cells in an efficient and accurate manner. In this proposal, we address this key issue by proposing a novel microengraving technology that allows unprecedented high-throughput testing of the infection status of individual primary cells. For this purpose, we will use a unique biochip that allows for the physical separation of individual mononuclear cells into more than ~105-106 wells with subnanoliter volumes. Protein microarrays printed from the supernatant of each individual cell and their subsequent interrogation with gp120 antibodies will then be used to detect cells with active viral production on a single cell level. By selectively subjecting primary activated cells or quiescent cells following ex vivo activation to this assay, we will be able to identify, physically isolate, and expand lytically and latently HIV-1 infected cells, and to study their key biological properties, including their TCR repertoire and clonotypic composition, their phenotypic characteristics, and the molecular mechanisms that determine their growth, proliferation and long-term maintenance. Moreover, applying this novel, multiplexed technology to the analysis of mononuclear cells from peripheral blood and tissue specimens in individuals with different rates of HIV-1 disease progression will allow us to determine specific quantitative or qualitative characteristics of the lytic or latent HIV-1 reservoir associated with distinct clinical outcomes of HIV-1 infection. This project is a collaboration between the Love lab (MIT) with expertise in engineering of single-cell biochip assays and the Yu lab (Ragon Institute/MGH) with expertise in the immunology and virology of HIV. This interdisciplinary approach to one of the most significant problems in HIV-1 research has the potential to result in novel strategies to target latently infected cells, and thus to cure HIV-1 entirely. PUBLIC HEALTH RELEVANCE: Quiescent CD4 T cells with latent HIV-1 infection represent the main reason for viral persistence and our inability to cure HIV-1 infection. In this proposal, we will use a novel, multiplexed bio-chip analysis system that will allow to identify, isolate and characterize these latently infected cells, and to contribute to the development of specific therapeutic strategies to target them.