Inducible HIV RNA transcription assays to measure HIV persistence: pros and cons of a compromise

Abstract

With the increasing number of therapeutic strategies tested in humans to reduce the size of the latent reservoir, the development of a robust, precise and clinical trial scalable assay that measures the frequency of infected cells carrying inducible replication-competent HIV is urgently needed. The size of the pool of cells carrying replication-competent HIV is largely overestimated by DNA assays, as a result of a large proportion of defective viruses, and underestimated by co-culture outgrowth assays. New culture methods that measure the inducible HIV reservoir have been developed during the past few years. In these induction assays, CD4+ T cells from virally suppressed individuals are activated and HIV RNA is measured in cell extracts or cell supernatants. In this review, we summarize the principle and outcomes of these assays and discuss the potential of these methods in the evaluation of HIV eradication strategies.

Improved assays to measure and characterize the inducible HIV reservoir

“…2 B). Only a median of 25% [ [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] ] of all positive wells were positive for all three RNA transcripts ( Fig. 2 B), suggesting that the majority of cells producing ca-RNA are unable to produce viral particles.…”

“…2 B), suggesting that the majority of cells producing ca-RNA are unable to produce viral particles. Some wells were positive for one of the three transcripts only (27% [ [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] ] gag ca-RNA only, 0% [0–3] tat-rev ca-RNA only, and 4% [ [2] , [3] , [4] , [5] , [6] ] cf-RNA only), which could be explained by different activation status of the infected cells at the time of nucleic acid extraction, distinct expression kinetics of the different transcripts, or deletions or other proviral sequence variation.…”

“…3 A). The frequencies of the inducible reservoir from 19 subjects were 94 [60–132] for gag and 16 [ [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] ] for tat-rev ca-RNA cells/10 6 CD4 + T cells, which were 29 and 4.9 times higher, respectively, than the frequencies of replication competent virus measured by mQVOA (p < .0001 and 0.009, respectively). However, they were 6.8 and 40 times lower, respectively, than the frequency of cells harboring total HIV DNA (p < .0001 in both cases).…”

“…Therefore, it is necessary to improve current assays to be able to detect precisely a diminution in the size of the HIV reservoir after a cure intervention before considering treatment interruption [ 33 ]. A large number of assays have been developed to quantify the frequency of HIV infected cells that persist in ART-suppressed individuals [ 11 , 19 , 22 , 23 , 34 , 35 ]. Here, we describe several assays that could be used to improve the sensitivity and dynamic range of HIV reservoir measurements.…”

“…Recently, assays of HIV RNA transcripts induced from CD4 + lymphocytes have been developed to measure the inducible HIV reservoir [ [21] , [22] , [23] ]. These assays have the advantage of reducing the contribution of most defective genomes and not relying on an amplification step with virus propagation by co-culture.…”