Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1

Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy 1. Current research efforts to cure HIV-1 infection include “shock and kill” strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells 2. However, the modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy 3,4. Aminobisphosphonates that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies 5. Here, we show the use of aminobisphosphonates as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that aminobisphosphonates induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin. RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further of alendronate-mediated latency reversal and activation of immune effector cells.Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of aminobisphosphonates to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination. Resting CD4 (rCD4) T cells were magnetically isolated from peripheral blood mononuclear cells (PMBC) from virally suppressed people living with HIV. All participants provided informed consent under an IRB approved study protocol. Cells were exposed to 2.5ug/mL pamidronate for six hours and plated untreated cells were used as a control. Cells were then harvested, pelleted and stored at -80. RNA was isolated using the automated KingFisher (Thermofisher Scientific, Waltham, MA). All RNA samples were assayed for integrity, concentration, and fragment size on a TapeStation system (Agilent, Inc. Santa Clara, CA). Gene expression profiles from rCD4T cells were generated by RNA-sequencing using an Illumina HiSeq. Library was constructed using Truseq RNA Exome library construction following the steps: Fragmenting RNA, Synthesizing 1st Strand cDNA, Synthesizing 2nd Strand cDNA, Adenylating 3' Ends, Ligating Adaptors, Enriching DNA fragments, Hybridizing Probes to library, capturing Hybridized Probes, performing a 2nd Hybridization, performing a 2nd capture, Clean-up of captured library was performed via AMPure XP Beads followed by amplification of the enriched library, and a final clean-up of amplified enriched library using AMPureXP beads. Libraries were prepared on an Agilent Bravo Automated Liquid Handling System. Indexed libraries were prepared and run on HiSeq4000 to generated paired end 75 base pair reads which generated approximately 90 million reads per sample library with a target of greater than 90% mapped reads.