Defining and then Maximizing the Footprint of CTL Selection on HIV Integration Site Landscapes to Optimize Cure Strategies - PROJECT SUMARY/ABSTRACT Eradicating HIV from infected individuals is obstructed by the formation of a reservoir of persistent infected cells; eliminating this reservoir could allow a cure for HIV infection. Recent research provides clues to approaches for reservoir eradication. Rare, infected individuals, termed elite controllers (EC), control HIV without treatment with antiretroviral medications. Analysis of HIV reservoirs in these individuals supports a model in which most efficiently expressed HIV proviruses have been eliminated. Thus, integration sites of proviruses from ECs are found enriched in genomic regions associated with heterochromatin and transcriptional repression. This overall pattern is, however, violated by some clonally expanded infected cells with genic integrations that show ongoing viral transcription. It has been suggested that these clones may have pro-survival and/or immune resistance characteristics, but this remains poorly understood. Our team has shown that the above features can be recapitulated by the application of CD8+ T-cell pressure in a novel mouse model of long-term infection, termed the Participant Derived Xenograft (PDX) model. For this, T-cells are obtained from ECs and separated into fractions. Naïve T cells are removed, which eliminates graft-versus-host disease upon transplantation into mice. CD4+ memory T cells are transplanted into immunodeficient mice, then HIV is introduced. Autologous CD8+ T- cells are introduced or not in controls, allowing experimental assessment of CD8+ T-cell pressure. In preliminary data, in the presence of CD8+ T-cells, the selected proviral population is smaller and viral loads lower. Advanced sequencing of integration site distributions shows larger clone sizes in the presence of CD8+ T-cell pressure, and multiple features paralleling results in ECs, such as favored integration outside transcription units, integration in reverse orientation relative to host transcription, and favored integration in more heterochromatic nuclear compartments to name a few. Thus, we propose to use the PDX model, advanced integration site analysis, and further tools to address the following Specific Aims: Aim 1. To define in high resolution the integration site features that enable the persistence of HIV proviruses under extended in vivo selection by CD8+ T-cells and compare the effects of diverse approach to enhancing this pressure. We will test multiple interventions in the PDX model to increase CD8+ T-cell pressure, reverse latency or block its establishment, and characterize in detail the molecular correlates. Aim 2. To train and validate multivariate statistical models to quantify the degree of CD8+ T-cell selection on a given pro-viral landscape and use the models to infer selection in interventional clinical trials. Completion of this study will thus provide advanced tools for quantifying CD8+ T-cell pressure on populations of integrated HIV proviruses, rich information on the functions of latency and immune modulators in the PDX model and in human trials, and an evaluation of the novel hypothesis that HIV insertional mutagenesis can confer resistance to CD8+ T-cell-mediated killing.
