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A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

ABSTRACT: Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4+ T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4+ T cells from whole PBMC is expedited and achieved in 3?hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.

SUBMITTER: Fun A

PROVIDER: S-EPMC5324126 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

Fun Axel A Mok Hoi Ping HP Wills Mark R MR Lever Andrew M AM

Scientific reports 20170224

Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in re ...[more]

PMID: 28233807

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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

A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

Publications

A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

Similar Datasets

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