Quantification of HIV-1 latency reversal in resting CD4+ T cells from patients on suppressive antiretroviral therapy

Abstract

Reversal of proviral latency is being pursued as a curative strategy for HIV-1 infection. Recent clinical studies of in vivo administration of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) show increases in unspliced cellular HIV-1 RNA levels in resting CD4(+) T cells. A critical unknown, however, is the proportion of latent proviruses that can be transcriptionally reactivated by SAHA or T-cell activation. In this study, we quantified the fraction of HIV-1 proviruses in resting CD4(+) T cells from patients on suppressive antiretroviral therapy that were reactivated ex vivo with SAHA or antibodies to CD3/CD28. At concentrations of SAHA achieved clinically, only 0.079% of proviruses in resting CD4(+) T cells were reactivated to produce virions, compared with 1.5% of proviruses in cells treated with anti-CD3/CD28 antibodies after correcting for spontaneous virion production in the medium control. A significant positive correlation (ρ = 0.67, P < 0.001) was found between levels of virions in the supernatant and unspliced cellular HIV-1 RNA following anti-CD3/CD28 treatment, but not following SAHA treatment (ρ = 0.21, P = 0.99). These results reveal that the majority of HIV-1 proviruses are not reactivated by current therapeutic approaches and that more effective means of reversing proviral latency will likely be required to deplete HIV-1 reservoirs.

Keywords: HIV-1 cure; HIV-1 eradication; HIV-1 persistence; fractional provirus expression.

Conflict of interest statement

Conflict of interest statement: J.W.M. is a consultant for Gilead Sciences and RFS Pharma and owns shares of RFS Pharma.

Figures

Fig. 1.

Illustration and explanation of the experimental system used to quantify fPVE. (A) Purified rCD4 cells are serially diluted and HIV-1 DNA copy number (per million rCD4 cells) is determined by qPCR to estimate the number of proviruses seeded per well. After treatment with a reactivating agent (or medium control), wells positive for HIV-1 virions in the supernatant are identified by RT-qPCR (Roche Taqman v2.0). The same experimental setup can be used to determine the fraction of proviruses that have been reactivated to produce cellular unspliced HIV-1 RNA by extracting total nucleic acid from cultured cells and quantifying HIV-1 RNA levels per well by RT-qPCR. (B) The number of positive wells from the hypothetical experiment in A was then tabulated, and a generalized linear model with a binomial distribution and a log link function was applied to determine the maximum likelihood estimate of fPVE. In the hypothetical example above, 1.5% of proviruses in rCD4 cells were reactivated to produce virions.

Fig. 2.

Only a small fraction of HIV-1 proviruses can be reactivated to produce virions during 7 d of culture with SAHA (0.5 μM) or anti-CD3/CD28 antibodies. The ratio of SAHA fPVE to anti-CD3/CD28 fPVE was 0.050 on average. Using a linear mixed effects model (34), anti-CD3/CD28 treatment significantly increased fPVE (P < 0.001), whereas neither medium control nor SAHA had a significant impact on fPVE. To determine mean fPVE values, the mean spontaneous fPVE (i.e., fPVE from medium control wells) was subtracted from the mean SAHA or anti-CD3/CD28 treated fPVE revealing that an average of 0.079% of proviruses can be reactivated to produce virions following SAHA treatment, and 1.5% of proviruses can be reactivated following anti-CD3/CD28 treatment. Open symbols represent samples below the limit of detection of fPVE for virion production. Symbols shown are values that fall outside 1.5 times the interquartile range of the boxplots.

Fig. 3.

Correlations between unspliced cellular HIV-1 RNA and virion production following treatment with anti-CD3/CD28 antibodies or SAHA. Virion-associated HIV-1 RNA levels were quantified from the supernatants of treated wells, and unspliced HIV-1 RNA was quantified from the cells in the same wells. Each data point is representative of one pair of virion and cellular RNA levels from one well. Several wells were selected at each dilution. (A) Treatment with anti-CD3/CD28 resulted in a significant positive correlation between unspliced cellular HIV-1 RNA and virion production in the same wells over 7 d of culture [ρ = 0.67, Spearman rank correlation based on resampling methods (30); P < 0.001, repeated measures analysis using generalized estimating equations]. (B) Treatment with SAHA led to unspliced cellular HIV-1 RNA production, but this increase in HIV-1 transcription did not correlate with virion production in the supernatant over 7 d [ρ = 0.21, Spearman rank correlation based on resampling methods (30); P = 0.99, repeated measures analysis using generalized estimating equations]. Negative values for virion-associated HIV-1 RNA and unspliced cellular HIV-1 RNA were set at 50% of the limit of quantification (10 copies and 1.5 copies, respectively, for each assay). Samples that were undetectable by either or both measures are shown as open symbols.