Combination antiretroviral therapy (ART) results in marked suppression of viremia in persons with HIV-1 infection. Therapy is not curative, however, & detectable viremia & replication-competent HIV-1 persist despite ART-induced suppression. The origin of persistent viremia on therapy is uncertain; potential sources include ongoing complete cycles of HIV-1 replication, long-lived reservoirs of chronically infected cells, sanctuary sites into which antiretrovirals have poor penetration, or a combination of these possibilities. Understanding the source & mechanisms of viral persistence on ART has critical implications for future therapeutic approaches & strategies for virus eradication. We began our investigation of the source of persistent HIV by developing an assay for viremia (HIV RNA) with single-copy sensitivity & by developing clinical protocols to determine the effects of ART intensification. These studies & others revealed no decrease in persistent viremia after drug intensification, suggesting that persistent viremia may be the product of long-lived reservoirs of chronically infected cells. Others have reported the utility of 2-LTR circles as an indicator of continued HIV replication during ART & increases in such circular forms during intensification with raltegravir. In addition, a survey of anatomic reservoirs revealed a higher HIV RNA to DNA ratio in cells from gut-associated lymphoid tissue (GALT) in the terminal ileum compared to the colon & rectum & decreases in HIV RNA during drug intensification. Such detailed analyses demonstrate the complexities of host-virus interactions & highlight the limitations in our understanding of mechanisms of persistence during suppressive ART.__The proposed project represents a focused approach to overcoming our limitations & understanding persistence by quantifying the host & viral contributions to HIV persistence. We are building on prior studies that used sensitive methodologies to quantify & genetically characterize virus in plasma to investigate HIV reservoirs in cellular compartments & are now expanding the range of our analyses by applying new single-cell methodologies & isolating specific cell subsets in blood & tissue from infected individuals.__To characterize the host-virus relationship during suppressive therapy, we are quantifying cellular & soluble immune correlates of persistent viremia. We initiated these studies by investigating the level of cellular immune activation before & after initiation of ART. The relative proportion of cellular immune activation markers (e.g., CD8+CD38+DR+cells) were high prior to therapy but declined sharply after ART was initiated; ultimately, HIV RNA levels & levels of immune activation stabilized to a persistent steady state with approximately the same time frame. Previous investigators detected persistent cellular immune activation during ART, but analyses to date have been restricted to 2-3 years on ART. To determine whether immune activation was still elevated after achieving steady-state persistent viremia, we quantified levels of cellular immune activation markers in patients with viremia suppressed for 7 years on ART & age-, sex- & race-matched uninfected controls. A modest but significant level of cellular immune activation (CD8+CD38+DR+ cells) was detectable even after 7 years on ART.__We investigated potential causes of persistent immune activation first by characterizing PBMC more fully, quantitating memory & naive, CD38, & DR subsets in CD4 & CD8 lineages. In parallel, we quantitated the levels of persistent viremia. Initial evaluation revealed a strong association between levels of persistent HIV RNA & CD8 memory subsets (r=0.51, p=0.0004) & CD8+CD38+DR+ immune activation (r=0.44, p=0.003). These data indicate that either generalized cellular activation itself drives production of HIV or activation is present in response to persistent viremia.__Determining the difference between these two possibilities will offer new insights for therapeutic strategies to eliminate such cellular reservoirs. If generalized activation is the source of persistent viremia, then treatment with agents that stimulate the immune system & increase cellular activation will result in increased HIV production from latent reservoirs, followed by overall decay in viremia. In contrast, if increased immune activation is directly controlling the level of persistent viremia, then further immune activation could result in its decay. With the HVIB Translational Research Unit, we will take a dual approach to define further characteristics of CD4+CD38+ & CD8+CD38+ cell subsets by quantifying additional markers in the long-term suppressed patients. Our prediction is that CD8 markers of activation & proliferation will correlate with viremia, but CD4 markers will not. If so, we will have identified a key distinction between cellular immune activation & viremia before & after introduction of ART.__We have now completed measurements of a series of soluble immune activation parameters (D-dimer, sCD14, hsCRP, IL-6, IP-10, sCD163, & are conducting an analysis of the relative levels of these markers, cellular immune activation parameters & single-copy assays._We are further characterizing the CD4 cell population by quantitating subsets of CD25+FoxP3+ (suppressor T reg) & IL-17+ (helper cells). Our hypothesis is that persistent viremia will be positively correlated with the relative frequency of FoxP3+ cells because these cells have immunosuppressive function, resulting in higher levels of HIV-1.__We are also using our well-characterized group of patients with long-term suppressed viremia on ART to characterize soluble markers of inflammation as correlates of viremia. Levels of soluble markers, such as D-Dimer, IL-6, C-reactive protein all cause mortality in HIV infection, even after suppression on ART.__There are no data correlating the relative levels of HIV viremia to predictive outcome markers. We will determine whether levels of soluble markers correlate with persistent viremia.__We will also quantify the effects of immune responses on HIV genetic variation. Using single-genome sequencing (SGS) techniques, we will determine whether prolonged viral suppression & partial immune reconstitution result in selection for cells infected with HIV immune escape variants. These studies will provide the first fine-structure analysis of HIV populations during prolonged ART.__In addition, we have initiated several new studies of HIV persistence. We are investigating HIV in plasma, PBMC, & cells derived from ileum & colon in infected individuals taking combination ART with suppressed 50 copies who are undergoing colonoscopy at the NIH Clinical Center. We have performed these colonoscopies in collaboration with J. Kovacs in the protocol Virologic & Immunologic Evaluation of Lymph Node, Tonsillar & Intestinal Biopsies, & Bronchoalveolar Lavage Fluid & have used a new sampling strategy that will yield useful information regarding the distribution of HIV-infected cells in the gastrointestinal tract. In a second study of HIV persistence, we are studying HIV from plasma & PBMC from patients with viral RNA suppressed on ART who undergo short antiretroviral discontinuation. Using SGS to investigate HIV from the earliest rebound viremia occurring within 7-14 days of discontinuation, we will identify a critical source of viremia.__In collaboration with S. Hughes (HIV DRP), X. Wu (Leidos), J. Coffin (Tufts), M. Kearney (HIV DRP) & J. Mellors (University of Pittsburgh), we have investigated HIV integration sites in vivo, characterizing HIV from plasma & PBMC of patients. Drs. Hughes & Wu have completed analysis of integration sites in these individuals. These studies revealed that specific integration sites may be linked to clonal expansion of HIV-infected cells, suggesting a novel mechanism for HIV persistence.
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