Comparison of methods to quantify inducible HIV-1 outgrowth (original) (raw)

Rapid Quantification of the Latent Reservoir for HIV-1 Using a Viral Outgrowth Assay

HIV-1 persists in infected individuals in a stable pool of resting CD4 + T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4 + T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4 + T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and costeffective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

A murine viral outgrowth assay to detect residual HIV-1 in patients with undetectable viral loads

The Journal of infectious diseases, 2015

Sensitive assays are needed for detection of residual HIV in patients with undetectable plasma viral loads to determine if eradication strategies are effective. The gold standard quantitative viral outgrowth assay (QVOA) underestimates the magnitude of the viral reservoir. We sought to determine whether xenograft of leukocytes from HIV-1 infected patients with undetectable plasma viral loads into immunocompromised mice would result in viral amplification. Peripheral blood mononuclear cells (PBMCs) or purified CD4+ T cells from HIV/SIV infected subjects with undetectable plasma viral loads were adoptively transferred into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. The mice were monitored for viremia following depletion of human CD8+ T cells to minimize antiviral activity. In some cases humanized mice were also treated with activating anti-CD3 antibody. With this murine viral outgrowth assay (MVOA), we successfully amplified replication-competent HIV-1/SIV from all subjects tested...

Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

PLoS pathogens, 2016

The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowt...

Quantitation of the latent HIV-1 reservoir from the sequence diversity in viral outgrowth assays

Retrovirology, 2018

The ability of HIV-1 to integrate into the genomes of quiescent host immune cells, establishing a long-lived latent viral reservoir (LVR), is the primary obstacle to curing these infections. Quantitative viral outgrowth assays (QVOAs) are the gold standard for estimating the size of the replication-competent HIV-1 LVR, measured by the number of infectious units per million (IUPM) cells. QVOAs are time-consuming because they rely on culturing replicate wells to amplify the production of virus antigen or nucleic acid to reproducibly detectable levels. Sequence analysis can reduce the required number of culture wells because the virus genetic diversity within the LVR provides an internal replication and dilution series. Here we develop a Bayesian method to jointly estimate the IUPM and variant frequencies (a measure of clonality) from the sequence diversity of QVOAs. Using simulation experiments, we find our Bayesian approach confers significantly greater accuracy over current methods ...

Measuring the Success of HIV-1 Cure Strategies

Frontiers in Cellular and Infection Microbiology, 2020

HIV-1 eradication strategies aim to achieve viral remission in the absence of antiretroviral therapy (ART). The development of an HIV-1 cure remains challenging due to the latent reservoir (LR): long-lived CD4 T cells that harbor transcriptionally silent HIV-1 provirus. The LR is stable despite years of suppressive ART and is the source of rebound viremia following therapy interruption. Cure strategies such as "shock and kill" aim to eliminate or reduce the LR by reversing latency, exposing the infected cells to clearance via the immune response or the viral cytopathic effect. Alternative strategies include therapeutic vaccination, which aims to prime the immune response to facilitate control of the virus in the absence of ART. Despite promising advances, these strategies have been unable to significantly reduce the LR or increase the time to viral rebound but have provided invaluable insight in the field of HIV-1 eradication. The development and assessment of an HIV-1 cure requires robust assays that can measure the LR with sufficient sensitivity to detect changes that may occur following treatment. The viral outgrowth assay (VOA) is considered the gold standard method for LR quantification due to its ability to distinguish intact and defective provirus. However, the VOA is time consuming and resource intensive, therefore several alternative assays have been developed to bridge the gap between practicality and accuracy. Whilst a cure for HIV-1 infection remains elusive, recent advances in our understanding of the LR and methods for its eradication have offered renewed hope regarding achieving ART free viral remission.

Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies

PLoS Pathogens, 2013

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4 + T cells carrying latent but replicationcompetent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, wellvalidated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4 + T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4 + T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research. Citation: Eriksson S, Graf EH, Dahl V, Strain MC, Yukl SA, et al. (2013) Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies. PLoS Pathog 9(2): e1003174.

Novel assays to measure total cell-associated HIV-1 DNA and RNA

Journal of clinical microbiology, 2016

Although a number of PCR-based quantitative assays have been reported for measuring HIV-1 persistence on suppressive antiretroviral therapy (ART), a simple, sensitive and reproducible method is needed for application to large clinical trials. We developed novel, quantitative PCR assays of cell-associated (CA) HIV-1 DNA and RNA targeting a highly-conserved region in HIV-1 pol with sensitivity of 3-5 copies/million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMC) from 5 viremic patients and 20 patients on effective ART. Total and resting CD4+ T cells were isolated in a subset of patients and tested for comparison with PBMC. The estimated standard deviations including inter- and intra-assay variability of the assays were modest: 0.15 and 0.10 log10 copies/10(6) PBMC for CA HIV-1 DNA; and 0.40 and 0.19 for CA HIV-1 RNA, respectively. Testing of longitudinally obtained PBMC samples showed little variation in either viremic ...