Venetoclax, alone and in combination with the BH3 mimetic S63845, depletes HIV-1 latently infected cells and delays rebound in humanized mice

Abstract

HIV-1 persists indefinitely in people living with HIV (PLWH) on antiretroviral therapy (ART). If ART is stopped, the virus rapidly rebounds from long-lived latently infected cells. Using a humanized mouse model of HIV-1 infection and CD4⁺ T cells from PLWH on ART, we investigate whether antagonizing host pro-survival proteins can prime latent cells to die and facilitate HIV-1 clearance. Venetoclax, a pro-apoptotic inhibitor of Bcl-2, depletes total and intact HIV-1 DNA in CD4⁺ T cells from PLWH ex vivo. This venetoclax-sensitive population is enriched for cells with transcriptionally higher levels of pro-apoptotic BH3-only proteins. Furthermore, venetoclax delays viral rebound in a mouse model of persistent HIV-1 infection, and the combination of venetoclax with the Mcl-1 inhibitor S63845 achieves a longer delay in rebound compared with either intervention alone. Thus, selective inhibition of pro-survival proteins can induce death of HIV-1-infected cells that persist on ART, extending time to viral rebound.

Graphical abstract

Figure 1

Viral rebound was delayed after a 6 week course of venetoclax (A) Schematic timeline depicting HIV-1 infection, suppressive ART period, and the beginning of each venetoclax treatment cycle (red arrows; administered every weekday). (B) Plasma viral loads of individual hu-mice over the course of the experiment (n = 6–8 mice per group). Limit of detection (LOD) is indicated with a dotted horizontal line (2.3 log₁₀ RNA copies mL⁻¹). (C) Kaplan-Maier curve representing the time to viral rebound following ART interruption. (D) Peripheral CD4⁺ T cell counts over the course of the experiment. (E) Frequency of CD4⁺ T cells expressing HIV-1 msRNA constitutively (i.e., unstimulated) (black) and following 24-h maximal stimulation with PMA/ionomycin (red). For (E), all data points represent one individual hu-mouse except for gray points, which represent pooled CD4⁺ T cells from 2 animals. For (E), n = 2 independent experiments. p value for Kaplan-Maier curve (C) was calculated using a log-rank Mantel-Cox test. p values for (E) were calculated using Wilcoxon matched-pairs signed-rank test. p value for each time point in (D) was analyzed using an unpaired t test corrected for multiple comparisons with the Holm-Šìdák method.

Figure 2

A delay in viral rebound was not detected after 3 weeks of S63845 treatment (A) Schematic timeline depicting HIV-1 infection, suppressive ART period, and the dosing regimen of S63845 (blue arrows; twice weekly). (B) Plasma viral loads of individual hu-mice over the course of the experiment (n = 8 mice per group). LOD is indicated with a dotted horizontal line (2.3 log₁₀ RNA copies mL⁻¹). (C) Kaplan-Maier curve representing the time to viral rebound following ART interruption. (D) Peripheral CD4⁺ T cell counts over the course of the experiment. p value for Kaplan-Maier curve (C) was calculated using a log-rank Mantel-Cox test. p value for each time point in (D) was analyzed using an unpaired t test corrected for multiple comparisons with the Holm-Šìdák method.

Figure 3

Viral rebound was delayed after a 3 week combined course of venetoclax and S63845 (A) Schematic timeline depicting HIV-1 infection, suppressive ART, and the dosing regimen of venetoclax (red arrows; administered every weekday) and S63845 (blue arrows; twice weekly). (B) Plasma viral loads of individual hu-mice over the course of the experiment (n = 4–7 mice per group). LOD is indicated with a dotted horizontal line (2.3 log₁₀ RNA copies mL⁻¹). (C) Kaplan-Maier curve representing the time to viral rebound following ART interruption. (D) Peripheral CD4⁺ T cell counts over the course of the experiment. p value for Kaplan-Maier curve (C) was calculated using a log-rank Mantel-Cox test. p value for each time point in (D) was analyzed using an unpaired t test corrected for multiple comparisons with the Holm-Šìdák method. “845” = S63845.

Figure 4

CD4⁺ T cells from PLWH on ART that survive ex vivo venetoclax co-culture have reduced levels of total and intact HIV-1 pro-viral DNA and exhibit a less apoptotic transcriptional signature Total CD4⁺ T cells isolated from peripheral blood of PLWH on ART were co-cultured with venetoclax (VNX) or DMSO control for 24 h before washing and harvesting 24 h later for quantification of HIV-1 DNA. For flow cytometry analysis and RNA sequencing (RNA-seq), cells were harvested immediately following 24 h VNX co-culture. (A) Fold change in total integrated HIV-1 DNA per million CD4⁺ T cells relative to DMSO control (n = 6 donors). (B) Absolute frequency of total integrated HIV-1 DNA per million CD4⁺ T cells for the 100 nM dose. (C) Fold change in intact HIV-1 DNA per million CD4⁺ T cells (n = 11 donors). (D) Absolute frequency of intact HIV-1 DNA per million CD4⁺ T cells for the 100 nM dose. (E) Heatmap visualizing the normalized proportion of dead/dying Violet⁺ cells within stated CD4⁺ T cell subsets at different VNX concentrations (n = 5 donors). (F and G) Normalized expression values by VNX treatment group for (F) pro-death and (G) pro-survival human Bcl-2 family genes. NA, naive; CM, central memory; EM, effector memory; TM, transitional memory; TD, terminally differentiated. For (B) and (D), MFC, median fold change. p values for (A) and (C) were calculated using a one-sample Wilcoxon signed-rank test. p values for (B) and (D) were calculated using a Wilcoxon matched-pairs signed-rank test. Error bars in (A) and (C) show median ± 95% confidence interval (CI). Boxplots in (F) and (G) show median ± interquartile range. Each symbol represents a different donor.