CD32-RNA Co-localizes with HIV-RNA in CD3+ Cells Found within Gut Tissues from Viremic and ART-Suppressed Individuals.

BackgroundIdentifying biomarkers for cells harboring replication-competent HIV is a major research priority. Recently, there have been mixed reports addressing the possibility that CD32-expressing T cells are enriched for HIV. There is growing evidence that CD32 expression increases with cellular activation that may be related to, but not necessarily specific for, infection with HIV. However, the relationship of CD32 expression to HIV-infection in subtypes of tissue-resident leukocytes is unclear.MethodsFirst, we used duplex chromogenic in situ hybridization to identify cells actively transcribing RNA for both CD32 and HIV on human gut tissues. Then we performed multiplexed immunofluorescence and in situ hybridization (mIFISH) on sections from the same tissues to determine the phenotype of individual cells co-expressing HIV-RNA and CD32-RNA.ResultsHIV-RNA+ cells were more abundant in tissues from viremic individuals than in those receiving suppressive anti-retroviral therapy (ART). However, staining by both methods indicated that a higher proportion of HIV-RNA+ cells co-expressed CD32-RNA in ART-suppressed individuals than in those with viremia. The majority of HIV-RNA+ cells were CD3+.ConclusionsOur data suggest that the transcription of CD32-RNA is correlated with HIV transcriptional activity in CD3+ cells found within human gut tissue. Whether or not up-regulation of CD32-RNA is a direct result of HIV transcription or more global T-cell activation remains unclear

Real-Time Predictions of Reservoir Size and Rebound Time during Antiretroviral Therapy Interruption Trials for HIV

Monitoring the efficacy of novel reservoir-reducing treatments for HIV is challenging. The limited ability to sample and quantify latent infection means that supervised antiretroviral therapy (ART) interruption studies are generally required. Here we introduce a set of mathematical and statistical modeling tools to aid in the design and interpretation of ART-interruption trials. We show how the likely size of the remaining reservoir can be updated in real-time as patients continue off treatment, by combining the output of laboratory assays with insights from models of reservoir dynamics and rebound. We design an optimal schedule for viral load sampling during interruption, whereby the frequency of follow-up can be decreased as patients continue off ART without rebound. While this scheme can minimize costs when the chance of rebound between visits is low, we find that the reservoir will be almost completely reseeded before rebound is detected unless sampling occurs at least every two weeks and the most sensitive viral load assays are used. We use simulated data to predict the clinical trial size needed to estimate treatment effects in the face of highly variable patient outcomes and imperfect reservoir assays. Our findings suggest that large numbers of patients—between 40 and 150—will be necessary to reliably estimate the reservoir-reducing potential of a new therapy and to compare this across interventions. As an example, we apply these methods to the two “Boston patients”, recipients of allogeneic hematopoietic stem cell transplants who experienced large reductions in latent infection and underwent ART-interruption. We argue that the timing of viral rebound was not particularly surprising given the information available before treatment cessation. Additionally, we show how other clinical data can be used to estimate the relative contribution that remaining HIV+ cells in the recipient versus newly infected cells from the donor made to the residual reservoir that eventually caused rebound. Together, these tools will aid HIV researchers in the evaluating new potentially-curative strategies that target the latent reservoir

Nanostructured Optical Photonic Crystal Biosensor for HIV Viral Load Measurement

Detecting and quantifying biomarkers and viruses in biological samples have broad applications in early disease diagnosis and treatment monitoring. We have demonstrated a label-free optical sensing mechanism using nanostructured photonic crystals (PC) to capture and quantify intact viruses (HIV-1) from biologically relevant samples. The nanostructured surface of the PC biosensor resonantly reflects a narrow wavelength band during illumination with a broadband light source. Surface-adsorbed biotarget induces a shift in the resonant Peak Wavelength Value (PWV) that is detectable with <10 pm wavelength resolution, enabling detection of both biomolecular layers and small number of viruses that sparsely populate the transducer surface. We have successfully captured and detected HIV-1 in serum and phosphate buffered saline (PBS) samples with viral loads ranging from 104 to 108 copies/mL. The surface density of immobilized biomolecular layers used in the sensor functionalization process, including 3-mercaptopropyltrimethoxysilane (3-MPS), N-gamma-Maleimidobutyryl-oxysuccinimide ester (GMBS), NeutrAvidin, anti-gp120, and bovine serum albumin (BSA) were also quantified by the PC biosensor

Designing and Interpreting Limiting Dilution Assays: General Principles and Applications to the Latent Reservoir for Human Immunodeficiency Virus-1

Limiting dilution assays are widely used in infectious disease research. These assays are crucial for current human immunodeficiency virus (HIV)-1 cure research in particular. In this study, we offer new tools to help investigators design and analyze dilution assays based on their specific research needs. Limiting dilution assays are commonly used to measure the extent of infection, and in the context of HIV they represent an essential tool for studying latency and potential curative strategies. Yet standard assay designs may not discern whether an intervention reduces an already miniscule latent infection. This review addresses challenges arising in this setting and in the general use of dilution assays. We illustrate the major statistical method for estimating frequency of infectious units from assay results, and we offer an online tool for computing this estimate. We recommend a procedure for customizing assay design to achieve desired sensitivity and precision goals, subject to experimental constraints. We consider experiments in which no viral outgrowth is observed and explain how using alternatives to viral outgrowth may make measurement of HIV latency more efficient. Finally, we discuss how biological complications, such as probabilistic growth of small infections, alter interpretations of experimental results

HIV-1 persistence in CD4+ T cells with stem cell-like properties

Cellular HIV-1 reservoirs that persist despite antiretroviral treatment are incompletely defined. We show that during suppressive antiretroviral therapy, CD4+ T memory stem cells (TSCM) harbor high per-cell levels of HIV-1 DNA, and make increasing contributions to the total viral CD4+ T cell reservoir over time. Moreover, phylogenetic studies suggested long-term persistence of viral quasispecies in CD4+ TSCM cells. Thus, HIV-1 may exploit stem cell characteristics of cellular immune memory to promote long-term viral persistence

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens

A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisininbased combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nucleasebased gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bisaminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates

Prediction of cardiovascular outcomes with machine learning techniques: application to the Cardiovascular Outcomes in Renal Atherosclerotic Lesions (CORAL) study.

Background: Data derived from the Cardiovascular Outcomes in Renal Atherosclerotic Lesions (CORAL) study were analyzed in an effort to employ machine learning methods to predict the composite endpoint described in the original study. Methods: We identified 573 CORAL subjects with complete baseline data and the presence or absence of a composite endpoint for the study. These data were subjected to several models including a generalized linear (logistic-linear) model, support vector machine, decision tree, feed-forward neural network, and random forest, in an effort to attempt to predict the composite endpoint. The subjects were arbitrarily divided into training and testing subsets according to an 80%:20% distribution with various seeds. Prediction models were optimized within the CARET package of R. Results: The best performance of the different machine learning techniques was that of the random forest method which yielded a receiver operator curve (ROC) area of 68.1%±4.2% (mean ± SD) on the testing subset with ten different seed values used to separate training and testing subsets. The four most important variables in the random forest method were SBP, serum creatinine, glycosylated hemoglobin, and DBP. Each of these variables was also important in at least some of the other methods. The treatment assignment group was not consistently an important determinant in any of the models. Conclusion: Prediction of a composite cardiovascular outcome was difficult in the CORAL population, even when employing machine learning methods. Assignment to either the stenting or best medical therapy group did not serve as an important predictor of composite outcome. Clinical Trial Registration: ClinicalTrials.gov, NCT00081731

A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates