Project Summary: Despite the efficacy and clinical benefits of effective ART, a reservoir of cells harboring replication-competent (intact) proviruses usually leads to viral rebound within weeks of stopping ART. Although this HIV reservoir is generally considered to be latent, sensitive PCR-based single copy HIV RNA assays have revealed low-level viremia in individuals on ART, indicating that latency is not absolute. Several groups including ours have shown that the HIV reservoir is maintained by the proliferation of infected cells carrying replication-competent (intact) proviruses (termed repliclones). Previously we reported that low-level persistent viremia on ART can originate from large clones carrying intact proviruses (Simonetti, et al. PNAS 2016; Halvas, et al. J Clin Invest 2020). This important finding raises the question of how repliclones escape immune clearance mechanisms despite proviral expression as evidenced by virion production and viremia. We postulate that one or more possible mechanisms may be involved that will be investigated in three Specific Aims of the current proposal. These mechanisms include: i) transcriptional silence (latency) of most proviruses in a repliclone; ii) dysfunctional immune responses that fail to mediate repliclone killing; iii) mutated proviruses in repliclones that escape CTL or antibody-mediated effector functions; and iv) intrinsic resistance of the repliclone cells to CD8+ T-cell- and/or NK cell-mediated killing. In Aim 1, we will characterize repliclones in 10-15 donors on effective ART (Aim 1A), and then quantify the fraction of proviruses in each clone that are expressing unspliced and single spliced (env) HIV mRNA by our recently reported cell-associated HIV RNA and DNA single genome sequencing method (Aim 1B). In Aim 2, we will evaluate three possible mechanisms by which transcriptionally active repliclones evade clearance by CD8+ T-cells. These evaluations will include: i) assessing epitope escape of repliclone proviruses (Aim 2A.1), ii) evaluating ex vivo cytotoxic activity of autologous T-cell responses with confirmed ability to recognize cells infected with repliclone virus (Aim 2A.2) and iii) directly determining whether cells comprising repliclones are inherently resistant to CD8+ T-cell-mediated killing (Aim 2B). In Aim 3, we will evaluate the neutralization sensitivity of viruses from individual repliclones to autologous and broadly-neutralizing monoclonal antibodies (Aim 3A), and the killing of repliclones by antibody-dependent cellular cytotoxic (ADCC) (Aim 3B). We will leverage a suite of novel tools already developed to detect repliclones, examine proviral expression in single repliclone cells, detect humoral and cellular immune escape variants, and assess the sensitivity of repliclones to killing by autologous virus-specific CD8+ T-cell clones or by ADCC. The work will be carried out by an ongoing, productive, and complementary collaboration between the Halvas and Mellors virology ...