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Comparison of methods to quantify inducible HIV-1 outgrowth.

ABSTRACT: The quantitative viral outgrowth assay (qVOA) is the gold standard for measuring inducible, replication-competent HIV-1. Using MOLT4-R5 and SupT1-R5 cell lines instead of allogeneic blasts and HIV-1 RNA detection rather than p24 enzyme-immunoassay (EIA) has been proposed to improve the sensitivity of the qVOA. It is unclear, however, how these alternative approaches affect qVOA performance. We compared three qVOAs methods across 15 persons with HIV-1 on suppressive antiretroviral therapy and found that the MOLT4-R5 method yielded a significantly higher proportion of p24-positive wells (42%) than both the allogeneic blast (29%) and SupT1-R5 (32%) assays. Additionally, 5 of 7 qVOAs that were negative by p24 EIA showed viral outgrowth by HIV-1 RNA quantification (>10-fold increase within 7 days). These findings reveal the potential for underestimation of the latent, inducible reservoir by qVOA depending on the target cells used and the measure of viral outgrowth. Use of MOLT4-R5 cells with both p24 EIA and HIV-1 RNA to detect viral outgrowth was the most sensitive method.

SUBMITTER: Enick PN

PROVIDER: S-EPMC8176359 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Comparison of methods to quantify inducible HIV-1 outgrowth.

Enick P Nathan PN Brooker Joseph P JP Tumiotto Camille M CM Staines Brittany T BT Eron Joseph J JJ McMahon Deborah K DK Gandhi Rajesh T RT Mellors John W JW Sobolewski Michele D MD

Journal of virus eradication 20210517 2

The quantitative viral outgrowth assay (qVOA) is the gold standard for measuring inducible, replication-competent HIV-1. Using MOLT4-R5 and SupT1-R5 cell lines instead of allogeneic blasts and HIV-1 RNA detection rather than p24 enzyme-immunoassay (EIA) has been proposed to improve the sensitivity of the qVOA. It is unclear, however, how these alternative approaches affect qVOA performance. We compared three qVOAs methods across 15 persons with HIV-1 on suppressive antiretroviral therapy and fou ...[more]

PMID: 34136266

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Project description:Quantifying the inducible HIV reservoir provides an estimate of the frequency of quiescent HIV-infected cells in humans as well as in animal models, and can help ascertain the efficacy of latency reversing agents (LRAs). The quantitative viral outgrowth assay (QVOA) is used to measure inducible, replication competent HIV and generate estimations of reservoir size. However, traditional QVOA is time and labor intensive and requires large amounts of lymphocytes. Given the importance of reproducible and accurate assessment of both reservoir size and LRA activity in cure strategies, efforts to streamline the QVOA are of high priority. We developed a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay, with ultra-sensitive p24 readout, capable of femtogram detection of HIV p24 protein in contrast to the picogram limitations of traditional ELISA. For each DEVO assay, 8-12 × 106 resting CD4 + T cells from aviremic, ART-treated HIV + participants are plated in limiting dilution and maximally stimulated with PHA, IL-2 and uninfected allogeneic irradiated PBMC. CD8-depleted PHA blasts from an uninfected donor or HIV-permissive cells (e.g., Molt4/CCR5) are added to the cultures and virus allowed to amplify for 8-12 days. HIV p24 from culture supernatant is measured at day 8 by Simoa (single molecule array, ultra-sensitive p24 assay) confirmed at day 12, and infectious units per million CD4 + T cells (IUPM) are calculated using the maximum likelihood method. In all DEVO assays performed, HIV p24 was detected in the supernatant of cultures as early as 8 days post stimulation. Importantly, DEVO IUPM values at day 8 were comparable or higher than traditional QVOA IUPM values obtained at day 15. Interestingly, DEVO IUPM values were similar with or without the addition of allogeneic CD8-depleted target PHA blasts or HIV permissive cells traditionally used to expand virus. The DEVO assay uses fewer resting CD4 + T cells and provides an assessment of reservoir size in less time than standard QVOA. This assay offers a new platform to quantify replication competent HIV during limited cell availability. Other potential applications include evaluating LRA activity, and measuring clearance of infected cells during latency clearance assays.

Dataset Information

Comparison of methods to quantify inducible HIV-1 outgrowth.

Publications

Comparison of methods to quantify inducible HIV-1 outgrowth.

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