Limits...
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

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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4563128&req=5

f0015: 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.

Mentions: Data presented in Fig. 2, Fig. 3 were generated by analysing samples obtained from individuals who participated in a previous study aimed at comparing different assays to measure HIV persistence (Eriksson et al., 2013). Briefly, this study enrolled 30 subjects from two well-established cohorts at the University of California San Francisco (UCSF). Samples from 27 of these participants were used for the present study (17 from the SCOPE cohort and 10 from the OPTIONS cohort). All of the 27 subjects had at least 36 months of continuous ART at study entry with no regimen changes in the preceding 24 weeks and maintenance of plasma HIV RNA levels below the limit of detection of conventional assays for at least 36 months (intermittent isolated episodes of detectable low-level viremia were allowed). Primary or recent HIV infection was defined if one or several of these 3 criteria were met: (Palella et al., 1998) repeated plasma HIV RNA > 5000 copies/ml combined with a negative or indeterminate HIV antibody test; (Murray et al., 2014) seroconversion within 6 months of a documented negative HIV antibody test; (Garrigue et al., 2000) a history compatible with primary HIV infection (including no prior positive HIV antibody tests) and laboratory testing consistent with recent infection on the “detuned” antibody EIA. Characteristics of these participants can be found in Eriksson et al. (2013).


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)

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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4563128&req=5

f0015: 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.
Mentions: Data presented in Fig. 2, Fig. 3 were generated by analysing samples obtained from individuals who participated in a previous study aimed at comparing different assays to measure HIV persistence (Eriksson et al., 2013). Briefly, this study enrolled 30 subjects from two well-established cohorts at the University of California San Francisco (UCSF). Samples from 27 of these participants were used for the present study (17 from the SCOPE cohort and 10 from the OPTIONS cohort). All of the 27 subjects had at least 36 months of continuous ART at study entry with no regimen changes in the preceding 24 weeks and maintenance of plasma HIV RNA levels below the limit of detection of conventional assays for at least 36 months (intermittent isolated episodes of detectable low-level viremia were allowed). Primary or recent HIV infection was defined if one or several of these 3 criteria were met: (Palella et al., 1998) repeated plasma HIV RNA > 5000 copies/ml combined with a negative or indeterminate HIV antibody test; (Murray et al., 2014) seroconversion within 6 months of a documented negative HIV antibody test; (Garrigue et al., 2000) a history compatible with primary HIV infection (including no prior positive HIV antibody tests) and laboratory testing consistent with recent infection on the “detuned” antibody EIA. Characteristics of these participants can be found in Eriksson et al. (2013).

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