Improved assays are critical to better characterize the HIV reservoir and to reliably evaluate candidate intervention strategies. Here we describe different methods to quantify the HIV reservoir.

We developed an optimized quantitative viral outgrowth assay (QVOA) to quantify the frequency of cells harboring replication-competent HIV, which is simpler and more sensitive than classical QVOAs. We also developed new inducible RNA assays that concomitantly measure the frequency of cell-associated [ca-] (gag and tat-rev) and cell-free [cf-] HIV RNA after three days of anti-CD3/CD28 stimulation.

The median frequency of the infected cells measured after induction was 94 IQR[60–132], 16 IQR [9–29] and 2.9 IQR[1.9–6.8] cells/10⁶ CD4⁺ T-cells for ca-RNA gag and tat-rev, and cf-RNA, respectively. There are a large proportion of transcription-competent proviruses (ca-RNA) that seemed unable to form complete virions (cf-RNA), suggesting post-transcriptional blocks or defective proviruses. Importantly, the median frequency of infected CD4⁺ T-cells as estimated by 3-day inducible cf-RNA assay was not statistically different from the frequency measured by the QVOA (median of 3.3 [1.9–6.2] IUPM). The latently infected cells detected by the inducible cf-RNA assay correlated highly with the QVOA ( r= 0.67, p < .001), and both assays were equivalent in 60% of the samples tested, suggesting that most cells induced to produce virions are generating replication-competent virus.

These inducible RNA assays provide more sensitivity and a greater dynamic range for the monitoring of reduction of the reservoir by eradication strategies. Such assays may serve as robust and useful tools for clinical investigations of the HIV reservoir.