Combination antiretroviral therapy (ART) results in marked suppression of viremia in persons with HIV-1 infection. Therapy is not curative, however, and detectable viremia and 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 and mechanisms of viral persistence on antiretroviral therapy has critical implications for future therapeutic approaches and strategies for virus eradication. If persistent viremia is derived from complete cycles of HIV-1 replication, improving the potency and penetration of drugs that block new cycles of replication is essential to improving outcome. By contrast, if new cycles of viral replication are completely suppressed by current therapy and viremia is derived from reservoirs of long-lived chronically infected cells, new strategies targeting these reservoirs are necessary to cure infection. We began our investigation of the source of persistent HIV by developing an assay for viremia (HIV RNA) with single copy sensitivity and by developing clinical protocols to determine the effects of antiretroviral intensification. These studies and 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 therapy and increases in such circular forms during intensification with raltegravir. In addition, a survey of anatomic reservoirs revealed a higher HIV RNA to HIV DNA ratio in cells from gut associated lymphoid tissue (GALT) in the terminal ileum compared to the colon and rectum and decreases in HIV RNA during drug intensification. Such detailed analyses demonstrate the complexities of host virus interactions, and highlight the limitations in our understanding of mechanisms of persistence during suppressive ART. The proposed project represents a focused approach to overcoming our limitations and understanding persistence by quantifying the host and viral contributions to HIV persistence. We are building on prior studies that used sensitive methodologies to quantify and genetically characterize virus in plasma to now investigate HIV reservoirs in cellular compartments. We are expanding the range of our analyses by apply new single cell methodologies and isolating specific cell subsets in blood and tissue from infected individuals. To characterize the host virus relationship during suppressive therapy, we are quantifying cellular and soluble immune correlates of persistent viremia. We initiated these studies by investigating the level of cellular immune activation prior to and following initiation of ART suppression. 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 (data not shown);ultimately HIV RNA levels and levels of immune activation stabilized to a persistent steady state with approximately the same time frame. Previous investigators detected persistent cellular immune activation during suppressive therapy, but analyses to date have been restricted to 2-3 years on therapy. 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 more than7 years on ART and age-, sex-, and race-matched uninfected controls. A modest but significant level of cellular immune activation (CD8+CD38+DR+ cells) was detectable even after >7 years of suppressive therapy. We investigatedpotential causes of persistent immune activation first by characterizing PBMC more fully, quantitating memory and naive, CD38, and DR subsets in CD4 and CD8 lineages. In parallel, we quantitated the levels persistent viremi. Initial evaluation revealed a strong association between levels of persistent HIV RNA and CD8 memory subsets (r=0.51, p=0.0004) and CD8+CD38+DR+ immune activation (r=0.44, p=0.003, Figure 5). These data indicate that either generalized cellular activation itself drives production of HIV or that 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 and 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 HVIU Translational Research Unit we will take a dual approach to define further characteristics of CD4+CD38+ and CD8+CD38+ cell subsets by quantifying additional markers in the long term suppressed patients. Our prediction is that CD8 markers of activation and proliferation will correlate with viremia, but CD4 markers will not. If so, we will have identified a key distinction between cellular immune activation and viremia prior to and following introduction of suppressive therapy. We will also further characterize the CD4 cell population by quantitating subsets of CD25+FoxP3+ (suppressor T reg) and 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 also hypothesize that viremia will be inversely correlated with frequency of IL-17+ cells, which have helper function and therefore predicted to suppress viremia. In the process, we will investigate whether these markers are present in central, effector, or peripheral memory cells. We will also use 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 predict 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 are correlated with persistent viremia. We will also quantify the effects of immune responses on HIV genetic variation. Using single genome sequencing techniques, we will determine whether prolonged viral suppression and partial immune reconstitution results in selection for cells infected with HIV immune escape variants. These studies will provide the first fine structure analysis of HIV populations during prolonged suppressive therapy.This project corresponds to Project 1 in our Site Visit Report 2011
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