Dataset Information

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

ABSTRACT: Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4+ T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous-increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

SUBMITTER: Yucha RW

PROVIDER: S-EPMC5478213 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Publications

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

Yucha Robert W RW Hobbs Kristen S KS Hanhauser Emily E Hogan Louise E LE Nieves Wildaliz W Ozen Mehmet O MO Inci Fatih F York Vanessa V Gibson Erica A EA Thanh Cassandra C Shafiee Hadi H El Assal Rami R Kiselinova Maja M Robles Yvonne P YP Bae Helen H Leadabrand Kaitlyn S KS Wang ShuQi S Deeks Steven G SG Kuritzkes Daniel R DR Demirci Utkan U Henrich Timothy J TJ

EBioMedicine 20170504

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4<sup>+</sup> T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency ...[more]

PMID: 28529033

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Project description:Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10(-4) per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys.Annual HIV incidence, the number of newly infected individuals within a year, is the key measure of monitoring the epidemic's rise and decline. Developing reliable assays differentiating recent from chronic infections has been a long-standing quest in the HIV community. Over the past 15 years, these assays have traditionally measured various HIV-specific antibodies, but recent technological advancements have expanded the diversity of proposed accurate, user-friendly, and financially viable tools. Here we designed a high-throughput genomic HIV incidence assay based on the signature imprinted in the HIV gene sequence population. By combining next-generation sequencing techniques with bioinformatics analysis, we demonstrated that genomic fingerprints are capable of distinguishing recently infected patients from chronically infected patients with high precision. Our high-throughput platform is expected to allow us to process many patients' samples from a single experiment, permitting the assay to be cost-effective for routine surveillance.

Dataset Information

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

Publications

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

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