Measuring the latent reservoir in vivo

Massanella, Marta; Richman, Douglas D.

doi:10.1172/jci80567

Cited by 74 publications

(81 citation statements)

References 93 publications

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“…This concept underlies the quantitative viral outgrowth assay (QVOA) that is considered to be the "gold-standard" method for measuring the latent reservoir. The important problem of measuring the latent reservoir is discussed in detail by Marta Massanella and Douglas Richman in this issue (21). Longitudinal measurements by the QVOA in patients on suppressive ART established that the half-life of the pool of latently infected resting CD4 + T cells is extremely long (3.7 years), thus requiring over 70 years of treatment to eradicate a pool of 10 6 cells (9, 10).…”

Section: The Latent Reservoir As a Barrier To Curementioning

confidence: 99%

Recent developments in the effort to cure HIV infection: going beyond N = 1

Siliciano¹,

Siliciano²

2016

Journal of Clinical Investigation

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Section: The Latent Reservoir As a Barrier To Curementioning

confidence: 99%

Recent developments in the effort to cure HIV infection: going beyond N = 1

Siliciano¹,

Siliciano²

2016

Journal of Clinical Investigation

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“…The quantitative virus outgrowth assay (QVOA) represents the "gold standard" [4][5][6], but this assay underestimates the true size of the functional reservoir due to incomplete induction by PHA stimulation. Ho et al [7] showed that the functional HIV-1 reservoir may be 60-fold larger than originally estimated.…”

Section: Introductionmentioning

confidence: 99%

“…PCR based assays are also commonly used for quantifying the HIV-1 reservoir, but these assays overestimate the size of the functional reservoir because they cannot distinguish between replication-competent and defective viral genomes. Quantification of the HIV-1 reservoir by PCRbased methods typically give at least 100-fold higher numbers that the QVOA because of defective proviruses [4][5][6]. Many defective proviruses have large internal deletions [7,8].…”

Section: Introductionmentioning

confidence: 99%

Establishment and stability of the latent HIV-1 DNA reservoir

Brodin

Zanini

Thebo

et al. 2016

Preprint

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HIV-1 infection currently cannot be cured because the virus persists as integrated proviral DNA in long-lived cells despite years of suppressive antiretroviral therapy (ART). To characterize establishment, turnover, and evolution of viral DNA reservoirs we deep-sequenced the p17gag region of the HIV-1 genome from samples obtained from 10 patients after 3-18 years of suppressive ART. For each of these patients, whole genome deep-sequencing data of HIV-1 RNA populations before onset of ART were available from 6-12 longitudinal plasma samples spanning 5-8 years of untreated infection. This enabled a detailed analysis of the dynamics and origin of proviral DNA during ART. A median of 14% (range 0-42%) of the p17gag DNA sequences were overtly defective due to G-to-A hypermutation. The remaining sequences were remarkably similar to previously observed RNA sequences and showed no evidence of evolution over many years of suppressive ART. Most sequences from the DNA reservoirs were very similar to viruses actively replicating in plasma (RNA sequences) shortly before start of ART. The results do not support persistent HIV-1 replication as a mechanism to maintain the HIV-1 reservoir during suppressive therapy. Rather, the data indicate that viral DNA variants are turning over as long as patients are untreated and that suppressive ART halts this turnover.

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“…Replication-competent HIV-1 can be measured by QVOA. These terminal detected by an assay for either p24 antigen or HIV RNA in the supernatant of these 12 co-cultures over a two-to three-week period [8]. Each well is read as negative or 13 positive, which means that replication-competent virus was present in at least one of the 14 cells in the well.…”

mentioning

confidence: 99%

“…These challenges include: 1) QVOA requires that large volumes of blood be collected to 23 generate large numbers of Ficoll-purified peripheral blood mononuclear cells (PBMC), 24 which are generally further processed without freezing/thawing, into input resting CD4 + 25 T cells, 2) each assay takes weeks to complete and is expensive (∼$3,000), 3) substantial 26 personnel time is required for cell purification, culture and monitoring supernatants for 27 HIV replication, limiting test throughput to two to four QVOAs per lab per week, 4) 28 not all replication-competent virus is detected by a single QVOA [11], and 5) different 29 laboratories employ varying methods [8].…”

mentioning

confidence: 99%

Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size

Rosenbloom

Bacchetti

Stone

et al. 2018

Preprint

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Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4 + T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, PLOS 1/36. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/329672 doi: bioRxiv preprint first posted online Jun. 6, 2018; little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates -as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir -rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre-and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points. Author summaryThe latent reservoir of resting CD4 + T cells is a major, if not the primary, obstacle to curing HIV. Quantitative viral outgrowth assays (QVOAs) are used to measure the latent reservoir in ART-suppressed HIV-infected people. Using QVOA is difficult, however, as the fraction of cells constituting the latent reservoir is typically about one in one million, far lower than other infectious disease biomarkers. To study reliability of these assays, we distributed 75 PBMC samples from five ART-suppressed HIV-infected participants among four labs, each conducting QVOA and following prespecified sample batching procedures. Using a Bayesian statistical method, we analyzed detailed assay output to understand how results varied within bat...

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Measuring the latent reservoir in vivo

Cited by 74 publications

References 93 publications

Recent developments in the effort to cure HIV infection: going beyond N = 1

Recent developments in the effort to cure HIV infection: going beyond N = 1

Establishment and stability of the latent HIV-1 DNA reservoir

Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size

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