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. 2015 Jun 27;2(8):874-83.
doi: 10.1016/j.ebiom.2015.06.019. eCollection 2015 Aug.

A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals

Francesco Andrea Procopio  1 , Rémi Fromentin  1 , Deanna A Kulpa  1 , Jessica H Brehm  1 , Anne-Gaelle Bebin  1 , Matthew C Strain  2 , Douglas D Richman  2 , Una O'Doherty  3 , Sarah Palmer  4 , Frederick M Hecht  5 , Rebecca Hoh  5 , Richard J O Barnard  6 , Michael D Miller  6 , Daria J Hazuda  6 , Steven G Deeks  5 , Rafick-Pierre Sékaly  1 , Nicolas Chomont  1
Affiliations

A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals

Francesco Andrea Procopio et al. EBioMedicine. .

Abstract

Background: Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging.

Methods: We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.

Findings: In suppressed individuals on ART, we found the median frequency of latently infected CD4 + T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4 + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART.

Interpretations: Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation.

Research in context: In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

Keywords: Eradication; HIV; Inducible virus; Latency; Multiply spliced RNA; Reservoir; TILDA.

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Figures

Fig. 1
Fig. 1
TILDA can be used to measure the frequency of persistently infected cells in blood samples from virally suppressed subjects. (A) Principle of TILDA: PBMCs were isolated from 10–20 ml of blood and CD4 + T cells were enriched by negative magnetic selection. CD4 + T cells were precisely counted and directly distributed in a 96 well plate or stimulated for 12 h with PMA/ionomycin. Tat/rev msRNA were quantified by an ultrasensitive nested RT-PCR and the frequency of cells with inducible msRNA was determined using the maximum likelihood method. (B) Frequency of CD4 + T cells producing msRNA spontaneously (baseline, n = 19) or after 12 h of stimulation (induced, n = 20) in samples obtained from virally suppressed subjects. Horizontal bars indicate median values. For samples in which no positive cells were detected at baseline, the limit of detection (based on cell input) is plotted and represented as a crossed circle. P value was obtained from the Wilcoxon matched-pairs signed rank test. (C) Proportion of CD4 + T cells that produce msRNA spontaneously or after stimulation in virally suppressed subjects on ART. Only samples with detectable values at baseline (panel B) are represented.
Fig. 2
Fig. 2
TILDA correlates with several measures of HIV persistence. (A) Correlation between TILDA and the droplet digital PCR assay for HIV DNA in PBMCs. (B) Correlation between TILDA and the Alu-PCR assay for HIV DNA in PBMCs. (C) Correlation between TILDA and the droplet digital PCR assay for HIV DNA in rectal CD4 + T cells. (D) Correlation between TILDA and the droplet digital PCR assay for HIV DNA in resting CD4 + T cells. (E) Correlation between TILDA and the Alu-PCR assay for HIV DNA in resting CD4 + T cells. (F) Correlation between TILDA and single copy assay for residual viremia. (G) Correlation between TILDA and Q-VOA in resting CD4 + T cells. Black circles and grey circles denote subjects who started ART during chronic and early infection, respectively. P values were obtained from the Pearson (A, B, C, E, G) or Spearman test (D, F), according to the results of the normality test (see Methods).
Fig. 3
Fig. 3
Initiation of ART during early HIV infection leads to a restricted size of the reservoir measured by TILDA. The frequency of cells harbouring inducible msRNA was measured by TILDA on CD4 + T cells obtained from subjects who started ART during chronic (n = 17, red circles) or recent (n = 10, blue circles) infection. Horizontal bars indicate median values. P value was obtained from the Mann–Whitney test.
Fig. 4
Fig. 4
TILDA reveals that untreated HIV-infected individuals harbour a large pool of latently infected CD4 + T cells. (A) Frequency of CD4 + T cells producing tat/rev msRNA spontaneously (baseline) or after 12 h of stimulation (induced) in 13 samples obtained from viremic, untreated HIV-infected individuals. Horizontal bars indicate median values. P value was obtained from the Wilcoxon matched-pairs signed rank test. (B) Correlation between the frequency of CD4 + T cells spontaneously producing HIV msRNA and HIV plasma viremia. P value was obtained from the Pearson test. (C) Proportion of CD4 + T cells that produce tat/rev msRNA spontaneously or after stimulation in untreated HIV-infected subjects. (D) Pie charts show the average relative proportions of productively and latently infected CD4 + T cells in virally suppressed (ART) and untreated viremic (VIR) HIV-infected subjects. P value was obtained from the Mann–Whitney test. (E) Correlation between the frequency of CD4 + T cells producing HIV msRNA after stimulation and duration of HIV infection. P value was obtained from the Pearson test. (F) Frequency of CD4 + T cells producing HIV msRNA before and after stimulation in the presence (+ ARVs) or absence (− ARVs) of antiretrovirals (raltegravir, efavirenz and AZT). P values were obtained from the Wilcoxon matched-pairs signed rank test.
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