Combination of a Latency-Reversing Agent With a Smac Mimetic Minimizes Secondary HIV-1 Infection in vitro

Abstract

Latency-reversing agents (LRAs) are considered a potential tool to cure human immunodeficiency virus type 1 (HIV-1) infection, but when they are taken alone, virus production by reactivated cells and subsequent infection will occur. Hence, it is crucial to simultaneously take appropriate measures to prevent such secondary HIV-1 infection. In this regard, a strategy to minimize the production of infectious viruses from LRA-reactivated cells is worth pursuing. Here, we focused on a second mitochondria-derived activator of caspases (Smac) mimetic, birinapant, to induce apoptosis in latent HIV-1-infected cells. When birinapant was administered alone, it only slightly increased the expression of caspase-3. However, in combination with an LRA (e.g., PEP005), it strongly induced the expression of caspase-3 followed by enhanced apoptosis. Importantly, the combination eliminated reactivated cells and drastically reduced HIV-1 production. Finally, we found that birinapant decreased the mRNA expression of HIV-1 that was induced by PEP005 in the primary CD4+ T-cells from HIV-1-carrying patients as well. These results suggest that the combination of an LRA and an “apoptosis-inducing” agent, such as a Smac mimetic, is a possible treatment option to decrease HIV-1 reservoirs without the occurrence of HIV-1 production by reactivated cells.

A widely-distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro

“…The rationale behind using LRA as an HIV-1 cure is reactivating the latent HIV-1 provirus and inducing cell apoptosis via cytopathic effects or recognition by the host antiviral immunity 4, 7, 11, 12, 31 . We therefore investigated whether the experimental cure observed in the present study was indeed mediated by the reactivation of latent reservoirs.…”

“…Since antiviral cytotoxic T-lymphocytes (CTLs) and antibodies are absent in the Jurkat/NL system, latent HIV-1-infected cells reactivated by PEP005 would have been eliminated mainly by viral cytopathicity or cell apoptosis ( Supplementary Fig. 1c ) 10–12 . Therefore, we examined intracellular p24 levels and cell apoptosis during the early phase of drug treatment and observed an increase in p24 protein expression in cells treated with PEP005 (+/− EFdA) just 6 h after drug treatment initiation ( Fig.…”

“…As there are no antiviral CTLs and antibodies in the WIPE assay, elimination of reactivated cells is mostly due to viral cytopathicity or apoptosis of the reactivated cells. Notably, we recently reported that some LRAs, such as PEP005, strongly induce the upregulation of active caspase-3, resulting in enhanced apoptosis 12, 36 . Thus, the addition of an LRA appeared to successfully accelerate the elimination of latently HIV-1-infected cells in the WIPE assay.…”

“…The ratios of intracellular HIV-1 p24 + cells, GFP + cells, and the active form of caspase-3 expression were determined as previously described 9, 12, 36 . Briefly, Jurkat/NL or JLTRG/NL cells were washed twice with phosphate buffered salts (PBS) and stained with Ghost Dye Red 780 (TONBO Biosciences, San Diego, CA) for 30 min at 4°C.…”

“…LRAs reverse HIV-1 latency and induce viral production in cells latently infected with the virus. In theory, infected cells that express viral antigens are then killed by the human immune system, such as cytotoxic T lymphocytes, or viral cytopathic effects 10–12 . However, LRAs that appear potent in in vitro assays are not necessarily effective in vivo because the viral reservoir situation is quite different in vitro and in vivo 13–16 .…”