Summary Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (~9 kb) HIV-1 proviral genomes and determines potential replication-competency through genetic characterization. FLIPS provides a genome-scale perspective which addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% proviruses as intact and potentially replication-competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells.Trial RegistrationClinicalTrials.gov NCT01365065

Memory T cells maintained a relatively constant HIV-1 DNA integrant pool that was genetically stable during long-term effective ART. These integrants appear to be maintained by cellular proliferation and longevity of infected cells, rather than by ongoing viral replication.

Objective: The contribution of HLA-DR+ memory CD4+ T cells to the HIV reservoir during prolonged antiretroviral therapy is unclear as these cells are commonly excluded when assessing for replication-competent HIV. To address this issue, we examined the distribution of genetically intact HIV DNA within HLA-DR− and HLA-DR+ memory CD4+ T cells and the RNA transcriptional profile of these cells during antiretroviral therapy. Design/methods: Full-length DNA sequencing was used to examine the HIV DNA landscape within HLA-DR+ and HLA-DR− memory CD4+ T cells. RNA quantification and sequencing was used to interrogate the relationship between HLA-DR status and HIV RNA transcription. Results: HLA-DR+ CD4+ T cells contained a high frequency of genetically intact HIV genomes, contributing over half of the genetically intact viral sequences to the reservoir. Expansions of genetically identical sequences were identified in all T-cell subsets, indicating that cellular proliferation maintains genetically intact and defective viral DNA during therapy. Intracellular HIV RNA levels in HLA-DR+ and HLA-DR− T cells were not statistically different by either long terminal repeat quantitative PCR quantification or single-genome RNA sequencing of the p6-RT region. Conclusion: The high proportion of intact viral DNA sequences in the proliferative HLA-DR+ subset suggests they are critical in maintaining HIV infection during effective therapy. As such, these cells should be included in any immune intervention targeting HIV during effective therapy.