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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 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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f0020: 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.

Mentions: Our results confirm that the latent HIV reservoir is established early in infection (Strain et al., 2005, Archin et al., 2012, Buzon et al., 2014, Ananworanich et al., 2012, Chomont et al., 2009). However, the relative proportion of productively and latently infected CD4 + T cells during untreated HIV infection is not known. As TILDA can measure the frequency of cells spontaneously producing msRNA as well as the frequency of cells that has the ability to do so upon maximal stimulation (Fig. 1), we used our assay to measure both frequencies in CD4 + T cells obtained from HIV-infected individuals who did not receive ART (Table 1). CD4 + T cells containing tat/rev transcripts at baseline were readily detected in all samples from viremic individuals (Fig. 4A). Overnight stimulation with PMA/ionomycin led to a significant increase in the frequency of CD4 + T cells producing msRNA (median fold increase of 7.4 in the frequency of cells with HIV msRNA, p = 0.0005). The frequency of cells spontaneously producing these transcripts was strongly correlated with plasma viral load (r = 0.81, p = 0.0014, Fig. 4B). Our calculations indicate that only a minor fraction (18.6%) of CD4 + T cells with inducible HIV genomes were spontaneously producing tat/rev transcripts in vivo (Fig. 4C), whereas the majority of infected cells (81.4%) were latently infected and produced msRNA only upon stimulation. Therefore, similar to virally suppressed subjects, ART-naïve individuals harbour a large pool of CD4 + T cells in which msRNA can be induced upon stimulation (Fig. 4D). The frequency of latently infected CD4 + T cells in untreated HIV-infected individuals was positively correlated with the duration of HIV infection (r = 0.71, p = 0.0097, Fig. 4E), suggesting that the size of the latent and inducible reservoir gradually increases in untreated viremic subjects, a consequence of continuous seeding of the HIV reservoir.


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 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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Related In: Results  -  Collection

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f0020: 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.
Mentions: Our results confirm that the latent HIV reservoir is established early in infection (Strain et al., 2005, Archin et al., 2012, Buzon et al., 2014, Ananworanich et al., 2012, Chomont et al., 2009). However, the relative proportion of productively and latently infected CD4 + T cells during untreated HIV infection is not known. As TILDA can measure the frequency of cells spontaneously producing msRNA as well as the frequency of cells that has the ability to do so upon maximal stimulation (Fig. 1), we used our assay to measure both frequencies in CD4 + T cells obtained from HIV-infected individuals who did not receive ART (Table 1). CD4 + T cells containing tat/rev transcripts at baseline were readily detected in all samples from viremic individuals (Fig. 4A). Overnight stimulation with PMA/ionomycin led to a significant increase in the frequency of CD4 + T cells producing msRNA (median fold increase of 7.4 in the frequency of cells with HIV msRNA, p = 0.0005). The frequency of cells spontaneously producing these transcripts was strongly correlated with plasma viral load (r = 0.81, p = 0.0014, Fig. 4B). Our calculations indicate that only a minor fraction (18.6%) of CD4 + T cells with inducible HIV genomes were spontaneously producing tat/rev transcripts in vivo (Fig. 4C), whereas the majority of infected cells (81.4%) were latently infected and produced msRNA only upon stimulation. Therefore, similar to virally suppressed subjects, ART-naïve individuals harbour a large pool of CD4 + T cells in which msRNA can be induced upon stimulation (Fig. 4D). The frequency of latently infected CD4 + T cells in untreated HIV-infected individuals was positively correlated with the duration of HIV infection (r = 0.71, p = 0.0097, Fig. 4E), suggesting that the size of the latent and inducible reservoir gradually increases in untreated viremic subjects, a consequence of continuous seeding of the HIV reservoir.

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