The ingenol-based protein kinase C agonist GSK445A is a potent inducer of HIV and SIV RNA transcription

Abstract

Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 μg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.

Fig 1. GSK445A induces cellular factors associated with HIV latency reversal.

Expressions of cell surface CD69 (A), intracellular p-NFκB (pS529) (B), intracellular pCDK9 (pS175) and (C) intracellular cyclin T1 (D) following stimulation with increasing doses of GSK445A were measured by flow cytometry in CD4⁺ T cells from 4 HIV-infected and virally suppressed individuals. Representative histograms (left panels) and dose response curves (right panels) are shown.

Fig 2. GSK445A induces HIV expression in CD4⁺ T cells from HIV-infected and virally suppressed individuals.

(A) Free HIV particles were measured in the culture supernatants of CD4⁺ T cells obtained from 5 HIV-infected and virally suppressed individuals exposed to increasing doses of GSK445A. (B) Cell-associated multiply spliced HIV RNA (Tat/Rev) were measured in CD4⁺ T cells obtained from 5 HIV-infected and virally suppressed individuals exposed to increasing doses of GSK445A. (C) Viral production in culture supernatants of CD4⁺ T cells obtained from 6 HIV-infected and virally suppressed individuals and stimulated without (NS) or with PMA/ionomycin or GSK445A (25 nM). (D) Frequency of CD4⁺ T cells obtained from 5 HIV-infected and virally suppressed individuals producing Tat/Rev HIV transcripts when stimulated without (NS) or with PMA/ionomycin or GSK445A (25 nM). Frequencies of Tat/Rev⁺ cells were measured by TILDA. P values in C and D were obtained using one-way ANOVA with Tukey’s multiple-comparisons test.

Fig 3. GSK445A synergistically enhances IFN-γ production in HIV-specific CD8⁺ T cells upon antigen stimulation.

(A) Representative HLA-A0201 HIV Gag tetramer staining after 6 days of A2-Gag-FK10 peptide stimulation with prior 30 minutes GSK445A pulse. Percentage of tetramer⁺ CD8⁺ T cells in total CD8⁺ T cells is shown. (B) Fold change of HIV-specific CD8⁺ T cell numbers relative to the cells cultured without peptide and prior GSK445A pulse (No peptide) from 9 PWH are shown. (C) CD69 expression on HIV-specific CD8⁺ T cells from 7 PWH after 6 hours of the peptide stimulation with prior GSK445A pulse. (D) Representative expression of IFN-γ in HLA-A0301 tetramer⁺ HIV-specific CD8⁺ T cells or total CD8⁺ T cells after 6 hours of peptide stimulation. Percentage of IFN-γ⁺ cells within HIV-specific CD8⁺ T cells or total CD8⁺ T cells are shown. (E) Percentage of IFN-γ⁺ cells and (F) expression levels of IFN-γ (MFI) within HIV-specific CD8⁺ T cells from 7 PWH. Differences among conditions were analyzed with Friedman test followed by Dunn’s Multiple Comparisons test. *P< 0.05; **P< 0.01; ***P< 0.001.

Fig 4. Effects of GSK445A on CD4⁺ T cell dynamics in SIV-naive RM.

(A) Mean (+SEM) GSK445A drug levels in plasma of RM (n = 5) following 4 sequential intravenous infusions of GSK445A at 5 μg/kg, 10 μg/kg, 20 μg/kg and 20 μg/kg at 14-day intervals. (B) IL-6 levels in plasma of RM (n = 5) following sequential infusions of GSK445A. (C) Mean (+SEM) absolute counts of CD4⁺ memory T cells and (D) CD4⁺ naive T cells in blood of RM (n = 5) following sequential infusions of GSK445A at 5 μg/kg, 10 μg/kg, 20 μg/kg, 20 μg/kg and 15 μg/kg at 14-day intervals. (E) Mean (+SEM) frequencies of Ki67⁺ CD4⁺ memory T cells and (F) Ki67⁺ CD4⁺ naive T cells in blood of RM (n = 5) following sequential infusions of GSK445A at 5 μg/kg, 10 μg/kg, 20 μg/kg, 20 μg/kg and 15 μg/kg at 14-day intervals. (G) Mean (+SEM) frequencies of CD69⁺ CD4⁺ memory T cells and (F) CD69⁺ CD4⁺ naive T cells in blood of RM (n = 5) following sequential infusions of GSK445A at 5 μg/kg, 10 μg/kg, 20 μg/kg, 20 μg/kg and 15 μg/kg at 14-day intervals.

Fig 5. Effects of GSK445A on CD4⁺ T cell dynamics in SIV-infected RM on ART.

(A) Plasma SIV RNA profiles for 4 RM following SIVmac239M infection, ART initiation on day 56 post-infection (pi) and GSK445A infusion starting day 298 pi. (B) Absolute counts of CD4⁺ T cells in blood following 3 infusions of GSK445A at 15 μg/kg. Note that the first 2 doses of GSK445A were administered over 5 minutes while the 3rd dose was administered over 20 minutes. (C) Representative flow cytometric analysis of RM RM25866 showing CD69 expression on CD4⁺ memory and naïve T cells in peripheral blood up to 24 hours following the third infusion of GSK445A at 15 μg/kg. (D) Frequencies of CD69⁺ CD4⁺ memory T cells (left panel) and CD69⁺ CD4⁺ naïve T cells (right panel) in blood following GSK445A infusion.

Fig 6. Effects of GSK445A on plasma viral loads.

Plasma viral loads profiles of 4 RM following 3 infusions of GSK445A at 15 μg/kg. Limit of detection was 15 RNA copies/ml of plasma.