Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC₅₀ = 4.5 nM) compared with vorinostat (VOR; EC₅₀ = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC₅₀ = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART. Combination antiretroviral therapy has greatly improved the clinical outcome of HIV infection treatment. However, latent viral reservoirs established primarily in memory CD4 T cells persist even after long periods of suppressive antiretroviral therapy, which hinders the ability to achieve a prolonged drug-free remission or a cure of the HIV infection. Activation of HIV expression from latent reservoirs is a part of proposed strategies that may potentially lead to virus elimination and ultimately cure of the infection. In this study, we show that romidepsin, a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, is a potent activator of HIV expression in an in vitro model of viral latency as well as ex vivo in resting and memory CD4 T cells isolated from HIV-infected patients with suppressed viremia. Importantly, the ex vivo activation of latent HIV occurred at romidepsin concentrations lower than those achieved in drug-treated lymphoma patients. In addition, romidepsin exhibited a more potent effect than other drugs in the same class that have already been shown to activate HIV expression in vivo. Together, these results support the clinical assessment of romidepsin in HIV-infected patients on suppressive antiretroviral therapy.

Resting CD4⁺ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8⁺ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8⁺ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8⁺ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8⁺ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8⁺ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam₃CSK₄. In contrast, we did not observe CD8⁺ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist ‘ALT-803’, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8⁺ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8⁺ T-cells in HIV eradication strategies. Although modern therapies have greatly improved the lives of HIV-positive people with access to care, a cure remains elusive. This leaves these individuals burdened by a lifelong commitment to medication, and fails to fully restore health. Curing infection would likely require therapies that combine the ability to force the virus out the ‘latent state’ in which it hides, with immune responses able to kill unmasked infected cells, the so called “shock and kill” strategy. A critical aspect of this strategy is identifying drugs that are effective at shocking virus out of latency, known as latency reversing agents. In this study, we took the novel approach of using CD8⁺ T-cells, immune cells responsible for killing infected cells, as biosensors able to detect the unmasking of latently-infected cells. Using this method, we screened a panel of potential latency reversing agents. We found that while a subset of these agents exposed infected cells to the immune system, others did not. Our results establish a new method for screening potential latency reversing agents, and support the prioritization of the agents that were shown to be effective for combination with CD8⁺ T-cells in shock and kill strategies aimed at curing HIV infection.

Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs. IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently maintained in reservoirs of latently infected cells. Consequently, lifelong therapy is required to maintain viral suppression. Ultimately, new therapies that specifically target and eliminate the latent HIV reservoir are needed. Toll-like receptor agonists are potent enhancers of innate antiviral immunity that can also improve the adaptive immune response. Here, we show that a highly selective TLR7 agonist, GS-9620, activated HIV from peripheral blood mononuclear cells isolated from HIV-infected individuals with suppressed infection. GS-9620 also improved immune effector functions that specifically targeted HIV-infected cells. Previously published studies on the compound in other chronic viral infections show that it can effectively induce immune activation at safe and tolerable clinical doses. Together, the results of these studies suggest that GS-9620 may be useful for treating HIV-infected individuals on suppressive antiretroviral therapy.

HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. Current HIV therapies prevent AIDS by dramatically reducing, but not eliminating, HIV infection. A reservoir of HIV-infected cells persists during long-term antiviral therapy, and individuals are at increased risk to develop non-AIDS illnesses, e.g., accelerated heart, bone, or kidney disease. Novel strategies are thus needed to safely kill HIV-infected cells and reduce or eliminate the HIV reservoir. An emerging strategy to kill HIV-infected cells involves antibodies (Abs) that bind the HIV envelope protein (Env). Env can distinguish HIV-infected cells from uninfected cells, and some Env-specific Abs can kill HIV-infected cells by recruiting immune cells, e.g., NK cells and macrophages. Here, we developed a strategy to kill HIV-infected cells that is complementary to Env-specific Abs. We designed and evaluated Dual-Affinity Re-Targeting (DART) molecules that incorporate Env-binding specificities with a CD3-binding specificity to recruit and activate cytotoxic T cells. We report that HIVxCD3 DARTs potently and selectively kill HIV-infected cells. Furthermore, HIV DARTs perturb resting and activated viral reservoirs in cells isolated from individuals on antiviral therapy. This novel strategy may be an important element of future antiviral therapies that target the HIV reservoir.

To measure molecular changes underlying pathogen adaptation, we generated a searchable dataset of more than 12,000 mass spectrometry events, corresponding to lipids and small molecules that constitute a lipidome for Mycobacterium tuberculosis . Iron is essential for M. tuberculosis survival, and the organism imports this metal using mycobactin and carboxymycobactin siderophores. Detection of an unexpected siderophore variant and deletions of genes for iron scavenging has led to a revised mycobactin biosynthesis model. An organism-wide search of the M. tuberculosis database for hypothetical compounds predicted by this model led to the discovery of two families of previously unknown lipids, designated monodeoxymycobactins and monodeoxycarboxymycobactins. These molecules suggest a revised biosynthetic model that alters the substrates and order of action of enzymes through the mycobactin biosynthetic pathway. We tested this model genetically by solving M. tuberculosis lipidomes after deletion of the iron-dependent regulator ( ideR ), mycobactin synthase B ( mbtB ), or mycobactin synthase G ( mbtG ). These studies show that deoxymycobactins are actively regulated during iron starvation, and also define essential roles of MbtG in converting deoxymycobactins to mycobactin and in promoting M. tuberculosis growth. Thus, lipidomics is an efficient discovery tool that informs genetic relationships, leading to a revised general model for the biosynthesis of these virulence-conferring siderophores.