Progress in the development of interventions to reduce levels of residual HIV in patients on ART is hampered, in part, by the lack of sensitive and reliable assays that can measure the functional reservoir size and track changes following latency reversing treatments. In addition, the heterogeneity of cellular characteristics of cells with latent HIV and their relative low frequencies confounds efforts to characterize underlying molecular mechanisms of viral latency in vivo. We propose to adapt a microfluidic-emulsion based, single cell assay system as 1) an assay that can quantify individual cells that are transcriptionally active at rest and after pharmacologic treatment and 2) an approach that permits the isolation and enrichment of HIV DNA containing cells for individual-cell-interrogation of infected cell responses to pharmacologic treatments intended to reverse viral latency. During the R21 phase of this proposal, we will develop a single cell Taqman assay based on the microfluidics/emulsion platform as a measure of the functional reservoir in CD4 T-cells isolated from subjects on long term suppressive ART, validate its performance characteristics and compare it to results from the gold standard measure of replication-competent reservoir size by terminal dilution co-culture. Concurrently, we will optimize a PCR-activated cell sorting (PACS) approach to recover total nucleic acids from cells that harbor HIV and those that do not and establish that these nucleic acids are suitable for targeted gene expression studies that quantitatively analyze single cell responses to pharmacologic agents for reversing viral latency. During the R33 phase of this proposal, we will test the single cell Taqman assay for its ability to predict two key consequences of HIV persistence: kinetics of viral rebound following treatment interruption and abnormal T-cell and macrophage activation during long term suppressive ART. The former is made possible by the availability of a unique and valuable sample set from the Swiss Spanish Interruption of Treatment study. Further, we will use the PACS system to identify and enrich HIV DNA+ cells from blood and GALT of patients on long term ART, collect the nucleic acid from individual single cells and perform targeted gene expression measurements to assess the magnitude of basal and post latency-reversal treatment viral RNA expression and their correlation with genes associated with T-cell stimulation, immune function, and inhibitory signaling to define cellular correlates of reversal or lack of reversal of HIV latency.
The small amount of HIV that remains in nearly everyone infected with HIV even after a decade or more of antiviral treatment results in serious health consequences and requires patients to stay on antiviral drugs for life. There is great interest in developing ways to get rid of this residual virus but efforts to do so will require better tools to measure and study it. In this proposal, we will use new technologies that allow the identification and study of single cells to learn more about what treatments might get rid of the residual virus.
|Li, Peilin; Kaiser, Philipp; Lampiris, Harry W et al. (2016) Stimulating the RIG-I pathway to kill cells in the latent HIV reservoir following viral reactivation. Nat Med 22:807-11|
|Wong, Joseph K; Yukl, Steven A (2016) Tissue reservoirs of HIV. Curr Opin HIV AIDS 11:362-70|
|Jiang, Guochun; Mendes, Erica A; Kaiser, Philipp et al. (2015) Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation. PLoS Pathog 11:e1005066|
|Jiang, Guochun; Mendes, Erica A; Kaiser, Philipp et al. (2014) Reactivation of HIV latency by a newly modified Ingenol derivative via protein kinase CÎ´-NF-ÎºB signaling. AIDS 28:1555-66|