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

Procopio FA, Fromentin R, Kulpa DA, Brehm JH, Bebin AG, Strain MC, Richman DD, O'Doherty U, Palmer S, Hecht FM, Hoh R, Barnard RJ, Miller MD, Hazuda DJ, Deeks SG, Sékaly RP, Chomont N - EBioMedicine (2015)

Bottom Line: 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.TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f0005: 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.

Mentions: We sought to develop an assay that detects tat/rev msRNA (adapted from Pasternak et al., 2008) in maximally activated CD4 + T cells, using a limiting dilution format, postulating that this method will allow us to measure the frequency of cells harbouring inducible viruses. We chose to activate the cells with phorbol 12-myristate 13-acetate (PMA), which has been shown to induce a rapid onset of increased transcription from the HIV LTR in latently infected cell lines (Li et al., 1991), induces higher levels of viral production than PHA (Tong-Starkesen et al., 1989) and activates rare T cell subsets (Wang et al., 2013). Ionomycin, a calcium ionophore, is added to trigger calcium release, a requirement for N-FAT signalling. PMA ionomycin has been shown to induce high levels of viral production in several primary cell models of HIV latency (Spina et al., 2013) as well as in CD4 + T cells from virally suppressed individuals (Bullen et al., 2014, Laird et al., 2014). After stimulation, cells are distributed in 24 replicate wells in a 96 well plate and a nested PCR amplifying tat/rev transcripts is directly performed without RNA extraction (Fig. 1A). Using the maximum likelihood method, the frequency of cells producing msRNA is calculated. TILDA can also be used to measure the frequency of CD4 + T cells that spontaneously produce msRNA ex vivo by omitting the stimulation step.


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

Procopio FA, Fromentin R, Kulpa DA, Brehm JH, Bebin AG, Strain MC, Richman DD, O'Doherty U, Palmer S, Hecht FM, Hoh R, Barnard RJ, Miller MD, Hazuda DJ, Deeks SG, Sékaly RP, Chomont N - EBioMedicine (2015)

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.
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Related In: Results  -  Collection

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f0005: 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.
Mentions: We sought to develop an assay that detects tat/rev msRNA (adapted from Pasternak et al., 2008) in maximally activated CD4 + T cells, using a limiting dilution format, postulating that this method will allow us to measure the frequency of cells harbouring inducible viruses. We chose to activate the cells with phorbol 12-myristate 13-acetate (PMA), which has been shown to induce a rapid onset of increased transcription from the HIV LTR in latently infected cell lines (Li et al., 1991), induces higher levels of viral production than PHA (Tong-Starkesen et al., 1989) and activates rare T cell subsets (Wang et al., 2013). Ionomycin, a calcium ionophore, is added to trigger calcium release, a requirement for N-FAT signalling. PMA ionomycin has been shown to induce high levels of viral production in several primary cell models of HIV latency (Spina et al., 2013) as well as in CD4 + T cells from virally suppressed individuals (Bullen et al., 2014, Laird et al., 2014). After stimulation, cells are distributed in 24 replicate wells in a 96 well plate and a nested PCR amplifying tat/rev transcripts is directly performed without RNA extraction (Fig. 1A). Using the maximum likelihood method, the frequency of cells producing msRNA is calculated. TILDA can also be used to measure the frequency of CD4 + T cells that spontaneously produce msRNA ex vivo by omitting the stimulation step.

Bottom Line: 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.TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.

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.

No MeSH data available.


Related in: MedlinePlus