Andrea S. Foulkes

Circulating Monocytes in HIV-1-Infected Viremic Subjects Exhibit an Antiapoptosis Gene Signature and Virus- and Host-Mediated Apoptosis Resistance

Mechanisms that may allow circulating monocytes to persist as CD4 T cells diminish in HIV-1 infection have not been investigated. We have characterized steady-state gene expression signatures in circulating monocytes from HIV-infected subjects and have identified a stable antiapoptosis gene signature comprised of 38 genes associated with p53, CD40L, TNF, and MAPK signaling networks. The significance of this gene signature is indicated by our demonstration of cadmium chloride- or Fas ligand-induced apoptosis resistance in circulating monocytes in contrast to increasing apoptosis in CD4 T cells from the same infected subjects. As potential mechanisms in vivo, we show that monocyte CCR5 binding by HIV-1 virus or agonist chemokines serves as independent viral and host modulators resulting in increased monocyte apoptosis resistance in vitro. We also show evidence for concordance between circulating monocyte apoptosis-related gene expression in HIV-1 infection in vivo and available datasets following viral infection or envelope exposure in monocyte-derived macrophages in vitro. The identification of in vivo gene expression associated with monocyte resistance to apoptosis is of relevance to AIDS pathogenesis since it would contribute to: 1) maintaining viability of infection targets and long-term reservoirs of HIV-1 infection in the monocyte/macrophage populations, and 2) protecting a cell subset critical to host survival despite sustained high viral replication.

Concurrent Measures Of Total And Integrated HIV DNA Monitor Reservoirs And Ongoing Replication In Eradication Trials

Objectives:Interest in targeting HIV reservoirs is fueling trials that may decrease reservoir size and/or induce viral replication. Therefore, we aimed to develop strategies to sensitively measure changes in these parameters in patients on and off antiretroviral therapy (ART). Achieving these goals may help evaluate the effects of future clinical trials. Design:To determine the relationship between measurements of total and integrated HIV DNA and their role as markers of reservoir size and ongoing replication, these parameters were measured during the first year of ART, during long-term effective ART, and during a clinical trial aimed at targeting reservoirs. Methods:Total and integrated HIV DNA were measured in patient samples using quantitative PCR techniques. CD4+T cell counts and plasma viremia were also monitored. Results:Unintegrated HIV DNA became undetectable during the first year of ART. Total and integrated HIV DNA levels were generally equal in well controlled patients on ART, and low-level plasma viremia correlated best with integration measures. Finally, patients who controlled plasma viremia (<400 copies/ml) during interferon-&agr; monotherapy exhibited a decrease in the level of integrated but not total HIV DNA and a rise in the ratio of total to integrated HIV DNA over time. Conclusion:Our findings suggest that appearance of unintegrated HIV DNA reflects residual HIV expression and de-novo reverse transcription, providing insight into the mechanism by which interferon-&agr; reduces the HIV reservoir. We conclude that concurrent measurements of total and integrated HIV DNA provide information regarding reservoir size and ongoing replication in trials targeting HIV.

Natural killer cell activation distinguishes Mycobacterium tuberculosis-mediated immune reconstitution syndrome from chronic HIV and HIV/MTB coinfection.

BackgroundWith increased access to antiretroviral treatment (ART), immune reconstitution inflammatory syndrome (IRIS) in Mycobacterium tuberculosis (MTB)–infected populations remains a clinical challenge. We studied a cross-sectional cohort of HIV-infected subjects in Johannesburg (South Africa) to help define the immune correlates that best distinguish IRIS from ongoing MTB cases. MethodsWe studied HIV+ subjects developing MTB-related unmasking tuberculosis-related immune reconstitution inflammatory syndrome (uTB-IRIS) after ART initiation; control groups were subjects with HIV and HIV/tuberculosis-coinfected subjects with comparable ART treatment. Testing was conducted with whole blood–based 4-color flow cytometry and plasma-based Luminex cytokine assessment. ResultsNatural killer cell activation, C-reactive protein, and interleukin 8 serum concentration were significantly higher in uTB-IRIS subjects compared with both control groups. In addition, all MTB-coinfected subjects, independent of clinical presentation, had higher neutrophils and T-cell activation, together with lower lymphocytes, CD4+ T-cell, and myeloid dendritic cell counts. Using conditional inference tree analysis, we show that elevated natural killer cell activation in combination with lymphocyte count characterizes the immunological profile of uTB-IRIS. ConclusionOur results support a role for innate immune effectors in the immunopathogenesis of unmasking MTB-related IRIS and identify new immune parameters defining this pathology.

Increased microbial translocation in ≤180 days old perinatally human immunodeficiency virus-positive infants as compared with human immunodeficiency virus-exposed uninfected infants of similar age

Background: The effect of early versus deferred antiretroviral treatment (ART) on plasma concentration of lipopolysaccharide (LPS) and host LPS-binding molecules in human immunodeficiency virus (HIV)-infected infants up to 1 year of age was investigated. Methods: We evaluated 54 perinatally HIV-infected and 22 HIV-exposed uninfected infants (controls) at the first and second semester of life. All HIV-infected infants had a baseline CD4 of ≥25%, participated in the Comprehensive International Program of Research on AIDS Children with HIV Early Antiretroviral Therapy trial in South Africa, and were randomized in the following groups: group 1 (n = 20), ART deferred until CD4 <25% or severe HIV disease; and group 2 (n = 34), ART initiation within 6 to 12 weeks of age. LPS, endotoxin-core antibodies, soluble CD14 (sCD14), and LPS-binding protein (LBP) were measured in cryopreserved plasma. T-cell activation was measured in fresh whole blood. Results: At the first semester, LPS concentration was higher in HIV-infected infants than in controls; sCD14, LBP, and T-cell activation were higher in group 1 than in group 2 and controls. Although LPS was not correlated with study variables, viral load was positively associated with sCD14, LBP, or endotoxin-core antibodies. At the second semester, LPS was not detectable and elevated host LPS-control molecules values were sustained in all groups and in conjunction with ART in all HIV-infected infants. Conclusions: Although plasma concentration of LPS was higher in perinatally HIV-infected infants 0 to 6 months of age than in controls independent of ART initiation strategy, concentration of LPS-control molecules was higher in infants with deferred ART, suggesting the presence of increased microbial translocation in HIV-infected infants with sustained early viral replication.

A guide to genome-wide association analysis and post-analytic interrogation

This tutorial is a learning resource that outlines the basic process and provides specific software tools for implementing a complete genome‐wide association analysis. Approaches to post‐analytic visualization and interrogation of potentially novel findings are also presented. Applications are illustrated using the free and open‐source R statistical computing and graphics software environment, Bioconductor software for bioinformatics and the UCSC Genome Browser. Complete genome‐wide association data on 1401 individuals across 861,473 typed single nucleotide polymorphisms from the PennCATH study of coronary artery disease are used for illustration. All data and code, as well as additional instructional resources, are publicly available through the Open Resources in Statistical Genomics project: http://www.stat-gen.org.

Prediction based classification for longitudinal biomarkers

Assessment of circulating CD4 count change over time in HIV-infected subjects on antiretroviral therapy (ART) is a central component of disease monitoring. The increasing number of HIV-infected subjects starting therapy and the limited capacity to support CD4 count testing within resource-limited settings have fueled interest in identifying correlates of CD4 count change such as total lymphocyte count, among others. The application of modeling techniques will be essential to this endeavor due to the typically non-linear CD4 trajectory over time and the multiple input variables necessary for capturing CD4 variability. We propose a prediction based classification approach that involves first stage modeling and subsequent classification based on clinically meaningful thresholds. This approach draws on existing analytical methods described in the receiver operating characteristic curve literature while presenting an extension for handling a continuous outcome. Application of this method to an independent test sample results in greater than 98% positive predictive value for CD4 count change. The prediction algorithm is derived based on a cohort of n = 270 HIV-1 infected individuals from the Royal Free Hospital, London who were followed for up to three years from initiation of ART. A test sample comprised of n = 72 individuals from Philadelphia and followed for a similar length of time is used for validation. Results suggest that this approach may be a useful tool for prioritizing limited laboratory resources for CD4 testing after subjects start antiretroviral therapy.

A Simple Test of Class-Level Genetic Association Can Reveal Novel Cardiometabolic Trait Loci

Background Characterizing the genetic determinants of complex diseases can be further augmented by incorporating knowledge of underlying structure or classifications of the genome, such as newly developed mappings of protein-coding genes, epigenetic marks, enhancer elements and non-coding RNAs. Methods We apply a simple class-level testing framework, termed Genetic Class Association Testing (GenCAT), to identify protein-coding gene association with 14 cardiometabolic (CMD) related traits across 6 publicly available genome wide association (GWA) meta-analysis data resources. GenCAT uses SNP-level meta-analysis test statistics across all SNPs within a class of elements, as well as the size of the class and its unique correlation structure, to determine if the class is statistically meaningful. The novelty of findings is evaluated through investigation of regional signals. A subset of findings are validated using recently updated, larger meta-analysis resources. A simulation study is presented to characterize overall performance with respect to power, control of family-wise error and computational efficiency. All analysis is performed using the GenCAT package, R version 3.2.1. Results We demonstrate that class-level testing complements the common first stage minP approach that involves individual SNP-level testing followed by post-hoc ascribing of statistically significant SNPs to genes and loci. GenCAT suggests 54 protein-coding genes at 41 distinct loci for the 13 CMD traits investigated in the discovery analysis, that are beyond the discoveries of minP alone. An additional application to biological pathways demonstrates flexibility in defining genetic classes. Conclusions We conclude that it would be prudent to include class-level testing as standard practice in GWA analysis. GenCAT, for example, can be used as a simple, complementary and efficient strategy for class-level testing that leverages existing data resources, requires only summary level data in the form of test statistics, and adds significant value with respect to its potential for identifying multiple novel and clinically relevant trait associations.

Deep RNA Sequencing Uncovers a Repertoire of Human Macrophage Long Intergenic Noncoding RNAs Modulated by Macrophage Activation and Associated With Cardiometabolic Diseases

Background Sustained and dysfunctional macrophage activation promotes inflammatory cardiometabolic disorders, but the role of long intergenic noncoding RNA (lincRNA) in human macrophage activation and cardiometabolic disorders is poorly defined. Through transcriptomics, bioinformatics, and selective functional studies, we sought to elucidate the lincRNA landscape of human macrophages. Methods and Results We used deep RNA sequencing to assemble the lincRNA transcriptome of human monocyte‐derived macrophages at rest and following stimulation with lipopolysaccharide and IFN‐γ (interferon γ) for M1 activation and IL‐4 (interleukin 4) for M2 activation. Through de novo assembly, we identified 2766 macrophage lincRNAs, including 861 that were previously unannotated. The majority (≈85%) was nonsyntenic or was syntenic but not annotated as expressed in mouse. Many macrophage lincRNAs demonstrated tissue‐enriched transcription patterns (21.5%) and enhancer‐like chromatin signatures (60.9%). Macrophage activation, particularly to the M1 phenotype, markedly altered the lincRNA expression profiles, revealing 96 lincRNAs differentially expressed, suggesting potential roles in regulating macrophage inflammatory functions. A subset of lincRNAs overlapped genomewide association study loci for cardiometabolic disorders. MacORIS (macrophage‐enriched obesity‐associated lincRNA serving as a repressor of IFN‐γ signaling), a macrophage‐enriched lincRNA not expressed in mouse macrophages, harbors variants associated with central obesity. Knockdown of MacORIS , which is located in the cytoplasm, enhanced IFN‐γ–induced JAK2 (Janus kinase 2) and STAT1 (signal transducer and activator of transcription 1) phosphorylation in THP‐1 macrophages, suggesting a potential role as a repressor of IFN‐γ signaling. Induced pluripotent stem cell–derived macrophages recapitulated the lincRNA transcriptome of human monocyte‐derived macrophages and provided a high‐fidelity model with which to study lincRNAs in human macrophage biology, particularly those not conserved in mouse. Conclusions High‐resolution transcriptomics identified lincRNAs that form part of the coordinated response during macrophage activation, including specific macrophage lincRNAs associated with human cardiometabolic disorders that modulate macrophage inflammatory functions.

Antiretroviral therapy interruptions result in loss of protective humoral immunity to neoantigens in HIV-infected individuals

Objective:Sustained antiretroviral therapy (ART)-mediated viral suppression restores responses to vaccination in HIV-1-infected individuals. As ART interruption occur frequently in resource-constrained settings, we studied their effects on the ability to mount humoral immune responses against a neoantigen. Design:Treatment-naive HIV-1-infected individuals were treated with stavudine, lamuvidine and lopinavir/ritonovir. Individuals who maintained viral load less than 50 copies/ml and CD4+ T-cell counts more than 450 cells/&mgr;l for 6 months received three doses of rabies vaccine, and were randomized to 72 weeks of continuous ART (arm 1) or sequential 2, 4 and 8-week ART interruptions (arm 2). An additional vaccine dose was administered at study end. Methods:Neutralizing antibody titers to rabies virus were assessed in plasma with a rapid fluorescent focus-inhibiting test. Results:The proportion of participants achieving protective (>0.5 IU/ml) antibody titer after vaccination was similar (arm 1 = 92%; arm 2 = 91%), but over time the cumulative proportion of observations with protective titer was greater in arm 1 than arm 2 (P = 0.0177). From week 26 after vaccination, antibody titers were lower in arm 2 than arm 1, and volunteers in arm 2 lost protective antibody titers at a greater rate (P = 0.0029). After boosting, 100% of arm 1 and 95% arm 2 volunteers achieved protective antibody titer. Conclusion:Our data indicate that individuals undergoing recurring ART interruption retain lower neutralizing antibody titers to a neoantigen, but maintain the ability to mount secondary responses upon boosting, suggesting that they might benefit from vaccine schedule intensification.

Genetic Association Studies

Recent technological advancements allowing for large-scale sequencing efforts present an exciting opportunity to uncover the genetic underpinnings of complex diseases. In an attempt to characterize these genetic contributors to disease, investigators have embarked in multitude on what are commonly referred to as population-based genetic association studies. These studies generally aim to relate genetic sequence information derived from unrelated individuals to a measure of disease progression or disease status. The field of genomics spans a wide array of research areas that involve the many stages of processing from genetic sequence information to protein products and ultimately the expression of a trait. The breadth of genomic investigations also includes studies of multiple organisms, ranging from bacteria to viruses to parasites to humans. In this chapter, two settings are described in which population-based genetic association studies have marked potential for uncovering disease etiology while elucidating new approaches for targeted, individualized therapeutic interventions: (1) complex disease association studies in humans; and (2) studies involving the Human Immunodeficiency Virus (HIV).