Mapping HIV reservoir establishment in human tonsil organoids

About This Grant

Project Summary Combination antiretroviral treatment has transformed HIV (Human immunodeficiency virus) infection from a fatal disease to a manageable chronic condition. It requires, however, regular antiretroviral medication. Attempts to cure HIV fall short because of the existence of long-lived HIV reservoirs comprised of cells harboring non- productive proviruses. While HIV is integrated into the genome of these cells, no viral proteins are expressed, leaving the cells indistinguishable from uninfected cells, preventing clearance by antiviral treatments or the immune system. My proposal seeks to define the properties and features of nonproductively infected cells circulating in the blood or residing in lymphoid tissues. My project will leverage a dual-reporter virus which allows for the physical separation of cells nonproductively infected with HIV from those that are productively infected and uninfected cells. Additionally, we have worked to develop methods of culturing and infecting lymphoid cells derived from human tonsils to mimic infection in tissues. Utilizing these model systems, we will work to understand cellular and viral factors which influence nonproductive HIV infection. In Specific Aim 1, I will utilize spectral flow cytometry to phenotype the cell types present in our tonsil organoid model, delineating the cells that harbor non-productive proviruses. We will compare these results to those found in blood derived CD4+ T cells and measure the impact of other bystander cell types on the relative frequency of productive to non-productive infections. We will use RNA sequencing to compare the transcriptomic landscapes of nonproductively infected cells to those of productively infected cells. In Specific Aim 2, we will utilize established and novel techniques to identify viral characteristics which correlate with nonproductive HIV infection. We will use digital PCR technologies to measure proviral intactness, and parallel sequencing techniques to identify HIV integration sites. These experiments will collectively identify host and viral factors which play a role in HIV latency establishment in relevant primary human cells and organoid model systems. This research will be supervised by Dr. Viviana Simon, a recognized leader in the field of virology. I will additionally be aided by esteemed collaborators both at our institution and beyond, who will assist with technical aspects as well as my development as a budding researcher. My fellowship proposal will generate new insights into the non-productive HIV reservoir, provide opportunities to learn advanced techniques both on the bench and in data analysis, and prepare me for a successful career as an independent academic researcher and mentor.