Novel assay reveals a large, inducible, replication-competent HIV-1 reservoir in resting CD4+ T cells

Abstract

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication-competent virus persist in resting CD4⁺ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that can accurately and rapidly quantify inducible, replication-competent latent HIV-1 from resting CD4⁺ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible, replication-competent latent HIV-1. This assay has several advantages over existing technology in that it (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive; and (iv) can be readily adapted into a high-throughput format. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.

Fig. 1. Sensitivity of TZM-bl cells to replication competent HIV-1

a) Acutely infected HIV-1^BaL-positive CD4⁺ T lymphocytes (30% infection frequency as determined by flow cytometry of intracellular p24) were serially diluted with uninfected CD8⁺ T cell- PBMC. 1×10⁵ cells from each serial dilution were added to 5×10⁴ TZM-bl cells in a 96-well plate. β-gal activity was measured by chemiluminesence 48 h later. The relative light units (RLU) for the control (460,000 +/− 56,000) were subtracted from each assay sample. b) Latently HIV-1 infected ACH-2 cells were treated with 100nM PMA, serially diluted with uninfected CD8⁺ T cell-depleted PBMC, and added to TZM-bl cells, as described above. c) Correlation between the TCID₅₀ for HIV-1^BaL and β-gal activity in the TZM-bl cells. TZM-bl cells were infected with HIV-1^BaL and β-gal activity was measured 48 h later. d) Correlation between the β-gal activity for HIV-1^BaL 48 h post infection and virus production (assessed by extracellular virion associated HIV-1 RNA 10 days post-infection) e) Detection of β-gal activity in the TZM-bl cells as a function of HIV-1_BaL TCID₅₀. We observed a linear relationship between β-gal activity and TCID₅₀ down to a TCID₅₀ of 0.001. f) Detection of extracellular virion-associated RNA as a function of HIV-1_BaL TCID₅₀. We observed a linear relationship between extracellular virion-associated RNA and TCID₅₀ down to a TCID₅₀ of 1. P values (cited in the text) for c, d, e and f were obtained using the Pearson test.

Fig. 2. TZM-bl cells are insensitive to infection by replication defective HIV-1

a) J-Lat clones 10.3, 9.2 and 8.4 were stimulated with PHA, serially diluted with uninfected CD8⁺ T cell-depleted PBMC and added to TZM-bl cells. β-gal activity was measured 48 h later. b) 8E5 cells were serially diluted with uninfected CD8⁺ T cell-depleted PBMC and added to TZM-bl cells. β-gal activity was measured 48 h later. c) Different p24 amounts of wild type (wt) HIV-1^LAI, and a mutant virus containing the L289K mutation in reverse transcriptase that renders the enzyme defective, were added to TZM-bL cells and β-gal activity was measured 48 h later. Statistical comparison of wild type versus mutant HIV-1 β-gal activity was performed using a Student’s T test (*, P < 0.05).

Fig 3. TZM-bl based assay (TZA) to quantify inducible replication competent HIV-1 from rCD4⁺ T cells

a) Schematic overview of the TZA, b) Statistical comparison of the IUPM values determined by TZA or Q-VOA using a parametric unpaired T test (P=0061). c) Statistical comparison of fractional HIV-1 provirus expression (fPVE) determined by TZA or Q-VOA using a parametric ratio paired T test (P=0.0006). d) Correlation between TZA and total HIV-1 DNA in rCD4⁺ T cells. e) Correlation between Q-VOA and total HIV-1 DNA in rCD4⁺ T cells. f) Correlation between TZA and intracellular HIV-1 gag-pol mRNA+ cells/10⁶ rCD4⁺ T cells. g) Correlation between TZA and Q-VOA. P values for all correlations were obtained using the Pearson test.

Fig. 4. Relationship between β-gal RLU signal or extracellular virion-associated HIV-1 RNA and the TCID₅₀ for HIV-1^BaL

The experimental set up was same as described for Fig. 1c, 1d, 1e and 1f. The average patient RLU (n = 12) in our study was 1.8 ×10⁶. This value closely approaches the cut-off for detection of virus production as assessed by extracellular HIV-1 RNA.