Year: 2017
Type: article
Abstract: Background: The quantitative viral outgrowth assay (QVOA) can provide a minimal but reproducible measure of the frequency of true latent HIV infection, reported as infectious units per million cells (IUPM).Such rare events however, require many cells to assay, and current assays are costly and slow.Furthermore, the QVOA underrepresents the frequency of replication-competent proviral infection, as no single ex vivo stimulation results in the outgrowth of all proviruses.However, the QVOA yields a reproducible assessment over time of the frequency of replication-competent HIV, a key measure of the efficacy of cure strategies.Thus any efforts to streamline this assay is of high priority.Methods: We have developed a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay which takes advantage of the recently developed SIMOA platform (Quanterix Inc), capable of femtogram detection of HIV p24 protein in contrast to the picogram limitations of traditional ELISA.8-10×106 purified resting CD4+ T cells from aviremic, ART-treated HIV+ participants were PHA stimulated in limiting dilution in a 96 well-format.CD8-depleted PHA blasts from an uninfected donor were added to half of the cultures and virus allowed to amplify for 8-12 days.P 24 from culture supernatant was measured by SIMOA.Results: In a total of 6 such assays performed, HIV p24 was detected in the supernatant of cultures as early as day 8 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 traditionally used to expand virus.Conclusions: The DEVO assay uses fewer resting CD4+ T cells, and may provide an assessment in a shorter time than standard QVOA.This assay offers a new platform to quantify replication competent HIV for a variety of applications, such as measuring the frequency of infection in situations where the number of cells available may be limited, evaluating latency reversal agent (LRA) activity, and measuring clearance of infected cells following the addition of autologous immune effectors.
Source: Journal of Virus Eradication
Institution: University of North Carolina at Chapel Hill
Language: English
Topic: HIV Research and Treatment
Subfield: Virology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,950
OpenAlex ID: W3041527082