Although effective against models of HIV latency, ‘shock and kill’ approaches aimed at reducing HIV reservoirs have not yet yielded this outcome in clinical trials, pointing to the need for novel and more potent therapeutic agents. 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt), an enhancer of STAT activation, increases occupancy of the transcription factor STAT5 on the HIV LTR thereby reactivating latent HIV in CD4+ T-cells. We have previously shown that HODHBt enhances IL-15 mediated reactivation in cells isolated from aviremic participants. STAT5 can also enhance CD8+ T-cell effector functions by transducing IL-15 signaling. Since HODHBt enhances STAT5 activation, we hypothesized that HODHBt would act synergistically with IL-15 to enhance cytotoxic function (GZMB release) of ex vivo HIV-specific CD8+ T-cells from ART treated individuals.
Granzyme B (GZMB) ELISpots were performed on PBMCs from 14 people living with HIV in the who had been on suppressive ART for an average of 10.9 years (range, 7.4-18). Cells were stimulated with peptide pools spanning Gag, Pol, Nef, or Env, CMVpp65, with or without 1 ng/ml IL-15 and/or 50 ug/ml HODHBt. Supernatants were collected and cytokine secretion was analyzed using the CorPlex Human Cytokine Panel 1 10-Plex array (IFN-?, IL-1b, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22 and TNF-?).
HIV-specific GZMB-releasing responses were enhanced by treatment with HODHBt in combination with IL-15, relative to medium only as follows: (mean/mean) Gag – 6.8-fold (p=0.005), Pol – 6.8-fold (p=0.005), Nef – 12.8-fold (p<0.001), and Env - 3.7-fold (p=0.438, ns). These were substantially increased relative to enhancements with IL-15 alone (median IL-15+HODHBt/median IL-15+DMSO) Gag - 2.4-fold (p = 0.0001), Pol - 1.8-fold, (p=0.0009), Nef - 4.3-fold, (p=0.0002), and Env - 2.6-fold, (p=0.0005). HODHBt alone did not increase background (no peptide) above IL-15 alone. Across all conditions and peptides, except CMV, GZMB was significantly positively correlated with IFN? (r= 0.89, p<0.0001) and IL22 (r=0.73, p<0.001).
In addition to its LRA potential, we show that HODHBt synergizes with IL-15 to markedly enhance HIV-specific cytotoxic T-cell responses in ex vivo PBMCs from ART-treated donors. Our results highlight that pharmacologic enhancement of IL-15 mediated STAT activation can be a therapeutic strategy with the potential to enhance both the ‘shock’ and the ‘kill’ components of strategies aimed at depleting HIV reservoirs.