The HIV Latency Reversal Agent HODHBt Enhances NK Cell Effector and Memory-Like Functions by Increasing Interleukin-15-Mediated STAT Activation.

Elimination of human immunodeficiency virus (HIV) reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is "shock and kill." This strategy is based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) acts as an LRA by increasing signal transducer and activator of transcription (STAT) factor activation mediated by interleukin-15 (IL-15) in cells isolated from aviremic participants. The IL-15 superagonist N-803 is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanisms of action by promoting the activation of STATs and have shown some promise in preclinical models directed toward HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in natural killer (NK) cells and the biological consequences associated with increased STAT activation in NK cell effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increases in the secretion of CXCL-10 and interferon gamma (IFN-γ) and the expression of cytotoxic proteins, including granzyme B, granzyme A, perforin, granulysin, FASL, and TRAIL. This increased cytotoxic profile results in increased cytotoxicity against HIV-infected cells and different tumor cell lines. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and thus, targeting this pathway could be further explored for HIV cure interventions. IMPORTANCE Several clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit, they have been crucial to elucidate hurdles that "shock and kill" strategies have to overcome to promote an effective reduction of the latent reservoir to lead to a cure. These hurdles include low reactivation potential mediated by LRAs, the negative influence of some LRAs on the activity of natural killer and effector CD8 T cells, an increased resistance to apoptosis of latently infected cells, and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of shock and kill strategies aimed at HIV eradication. Here, we show that the LRA HODHBt also improves IL-15-mediated NK cell effector and memory-like functions. As such, pharmacological enhancement of IL-15-mediated STAT activation can open new therapeutic avenues toward an HIV cure.

An Ultrasensitive p24 Assay to Measure HIV-1 in Diverse Biological Matrixes.

Assays to study HIV persistence are crucial to evaluate therapeutic strategies aimed toward an HIV cure. Several assays have been developed to date that rely on the measurement of nucleic acids. In recent years, the advancement of ultrasensitive technologies for the detection of proteins has improved our understanding of the role of translation-competent reservoirs in HIV persistence. In this chapter, we describe the development of an ultrasensitive p24 ELISA that uses planar array technology. This assay allows for the detection of HIV-1 p24 in the low fg/ml range in different biological matrixes, including cell lysates. This assay can be used to investigate the efficacy of latency reversing agents to reactivate HIV or to evaluate the persistence of translation-competent reservoirs in people living with HIV (PWH) in cells or diverse biological fluids.

The latency-reversing agent HODHBt synergizes with IL-15 to enhance cytotoxic function of HIV-specific T cells.

IL-15 is under clinical investigation toward the goal of curing HIV infection because of its abilities to reverse HIV latency and enhance immune effector function. However, increased potency through combination with other agents may be needed. 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhances IL-15-mediated latency reversal and NK cell function by increasing STAT5 activation. We hypothesized that HODHBt would also synergize with IL-15, via STAT5, to directly enhance HIV-specific cytotoxic T cell responses. We showed that ex vivo IL-15 + HODHBt treatment markedly enhanced HIV-specific granzyme B-releasing T cell responses in PBMCs from antiretroviral therapy-suppressed (ART-suppressed) donors. We also observed upregulation of antigen processing and presentation in CD4+ T cells and increased surface MHC-I. In ex vivo PBMCs, IL-15 + HODHBt was sufficient to reduce intact proviruses in 1 of 3 ART-suppressed donors. Our findings reveal the potential for second-generation IL-15 studies incorporating HODHBt-like therapeutics. Iterative studies layering on additional latency reversal or other agents are needed to achieve consistent ex vivo reservoir reductions.

IL-27 induces IFN/STAT1-dependent genes and enhances function of TIGIT+ HIVGag-specific T cells.

HIV-specific T cells have diminished effector function and fail to control/eliminate the virus. IL-27, a member of the IL-6/IL-12 cytokine superfamily has been shown to inhibit HIV replication. However, whether or not IL-27 can enhance HIV-specific T cell function is largely unknown. In the present manuscript, we investigated the role of IL-27 signaling in human T cells by evaluating the global transcriptional changes related to the function of HIV-specific T cells. We found that T cells from people living with HIV (PLWH), expressed higher levels of STAT1 leading to enhanced STAT1 activation upon IL-27 stimulation. Observed IL-27 induced transcriptional changes were associated with IFN/STAT1-dependent pathways in CD4 and CD8 T cells. Importantly, IL-27 dependent modulation of T-bet expression promoted IFNγ secretion by TIGIT+HIVGag-specific T cells. This new immunomodulatory effect of IL-27 on HIV-specific T cell function suggests its potential therapeutic use in cure strategies.

An ultrasensitive planar array p24 Gag ELISA to detect HIV-1 in diverse biological matrixes.

Human immunodeficiency virus-1 (HIV-1) persistence in the presence of antiretroviral therapy (ART) has halted the development of curative strategies. Measuring HIV persistence is complex due to the low frequency of cells containing virus in vivo. Most of the commercially available assays to date measure nucleic acid. These assays have the advantage of being highly sensitive and allow for the analysis of sequence diversity, intactness of the HIV genome or evaluation of diverse RNA species. However, these assays are limited in evaluating translational competent viral reservoirs. In here, we developed an ultrasensitive p24 ELISA that uses the Simoa planar array technology that can detect HIV-1 virions and HIV-1 infected cell with limit of detection similar to nucleic acid assays. Furthermore, the assay is optimized to measure very low levels of p24 in different biological fluids without a major loss of sensitivity or reproducibility. Our results demonstrate that the 'homebrew' planar p24 ELISA immunoassay is a broadly applicable new tool to evaluate HIV persistence in diverse biological fluids and cells.