BackgroundImproved 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.MethodsWe 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.FindingsThe 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.InterpretationThese 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.

We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with qPCR results ( = 0.7052; = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

Latently infected CD4 lymphocytes preclude cure of HIV infection, even with the most effective antiretroviral therapy. The replication competent latent HIV reservoir has been quantified with the terminal dilution quantitative viral outgrowth assay, which induces virus propagation in CD4 + T cell culture supernatants following cellular activation. Efforts to improve the sensitivity of this inefficient assay have introduced more sensitive p24 ELISA and RNA PCR based endpoints, but these more sensitive endpoints have raised the question whether they are measuring induced replication competent or defective virions. Here we performed parallel terminal dilution assays with CD4 lymphocytes from subjects effectively treated with antiretroviral therapy. An HIV integrase inhibitor was incorporated into one set of parallel cultures to compare the frequency of cells that can be induced to produce virions to those that produce virus that can propagate and amplify with co-culture in permissive cells. The majority of cells that can be induced to generate virus particles are producing replication competent virus, thus justifying more sensitive and faster assays of this reservoir. Electronic supplementary material The online version of this article (10.1186/s12977-019-0466-1) contains supplementary material, which is available to authorized users.