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. 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. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found in ex vivo assays using primary cells from ART-suppressed people living with HIV-1 that N-BPs induce HIV-1 from latency to levels that are comparable to the T cell activator phytohemagglutinin (PHA). 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 evidence 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 N-BPs 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.


INTRODUCTION
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). Despite the appealing nature of the theory behind the "shock and kill" strategy for HIV-1 cure, the latency reversing agents (LRAs) that have progressed to clinical trials to date have not achieved a sustained decrease of the viral reservoir despite demonstrating in vivo induction of HIV-1 RNA transcription or transient detectable plasma viremia (3,4). Both the limited availability of Federal Drug Agency (FDA)-approved LRA compounds and their modest success to date urges the HIV-1 cure field to identify additional drugs for pre-clinical and clinical testing.
FPP is essential for protein prenylation, a post-translational modification that consists of the transfer of FPP groups to proteins involved in cell signaling, including GTPases from the Ras family (7,8). Interestingly, statins that inhibit upstream of IPP production have been shown to inhibit HIV-1 viral replication (9)(10)(11). Upstream accumulation of IPP also leads to the exclusive activation of the more frequent subpopulation of circulating gamma delta () T cells, V2 T cells, that specifically recognize IPP in a T cell receptor (TCR)-dependent manner (12)(13)(14). V2 T cells are potent cytotoxic effectors against virally infected or malignant cells (15)(16)(17), and we previously 4 reported the capacity of ex vivo expanded V2 T cells to target and eliminate latently HIV-1infected resting CD4 T cells upon ex vivo latency reversal (18,19). In addition, activated V2 T cells have a critical adjuvant activity by inducing antigen-experienced and naïve CD8 T cell responses (20)(21)(22)(23)(24). We hypothesized that N-BPs exert a dual effect by inducing both HIV-1 reactivation and concomitant immune responses through activation of V2 T cell cytotoxic and adjuvant effects that could lead to elimination of HIV-1 reservoirs.
Here, we show that ex vivo exposure to N-BPs reverses HIV-1 latency most likely through the activator protein 1 (AP-1) signaling pathway. In vivo treatment of ART-suppressed people living with HIV (PLWH) with ALN induced viral reactivation although a significant reduction in the reservoir size was not detected in our small cohort. This study provides evidence of the novel use of N-BPs as LRAs and warrants further investigations with the specific objective to assess latency reversal and infected cell clearance in a larger cohort. 6 To confirm that N-BPs induced production of non-defective, replication-competent HIV-1, rCD4 T cells from PLWH on ART were used in QVOAs using a minimum of 13

Mechanism of action of N-BPs on viral reactivation
To explore pathways involved in N-BPs reversing HIV-1 latency, we performed RNA-seq on ex vivo untreated or 2.5g/mL PAM-treated paired samples from six ART-suppressed PLWH. To exclude changes in gene expression, due to toxicity, a dose-curve analysis of PAM toxicity was performed showing that the selected dose did not impact cell viability ( Supplementary Fig. 2).
Next, to confirm the functional effect of N-BPs at selected concentrations in our experiment, we analyzed whether protein prenylation of Ras was reduced upon N-BP exposure. Our results showed that treatment with PAM, Zol and ALN decreased the expression of active Ras (GTP-Ras) after overnight incubation by 3.1-fold, 4.7-fold and 6.6-fold compared to media only control, respectively, indicating inhibition of protein prenylation ( Figure 2A). RNA-seq analysis identified 1,799 genes differentially expressed between samples treated with PAM and untreated controls ( Figure 2B, Supplementary Table 2). We next tested for differentially expressed gene modules using Gene Set Variation Analysis and linear regression (GSVA, Supplementary Table 3) (30).
Consistent with the effect of N-BPs decreasing the production of cholesterol (31), the cholesterol synthesis pathway (GO_Regulation of cholesterol biosynthetic pathway) was decreased in the samples treated with PAM compared to media only control ( Supplementary Fig. 3), confirming the specificity of PAM and validating the RNA-seq data. The top differentially expressed pathway was the response to bacterial lipoprotein, and identified TLR2 as one of the genes upregulated upon PAM treatment (p= 4.6x10 -15 ; Figure 2C and Supplementary Table 3). To confirm the effect of PAM on this pathway, we performed time-course surface TLR2 expression studies by flow cytometry in isolated CD4 T cells. In accordance with our RNA-seq data (Supplementary Table   2), we detected an early upregulation of surface TLR2 expression after four and six hours of incubation, followed by a downregulation after overnight exposure to N-BPs ( Figure 2D). Next, 8 we used the Predicting Associated Transcription factors from annotated affinities (PASTAA) (32) software to predict transcription factors (TF) associated with PAM-mediated transcriptional changes. The top-8 TFs associated with the PAM-mediated transcriptional changes were NKX2-1, IRF-8, ETV4, POU2F1, two subunits of AP-1 (FOSB and JUN), ELF-4 and Cart-1. STRING pathway analysis of interactions of these TFs with the GTPases activated upon inhibition of protein prenylation (Rac, Cdc42 and Rho) revealed a unique cluster of these GTPases with JUN (c-Jun) ( Figure 2E), that has been previously shown to be involved in viral reactivation from latency (33,34). Since c-Jun transcriptional activation depends on phosphorylation at Ser73 of the transactivation domain for transcriptional activity (35) we next analyzed the phosphorylation status of c-Jun at this residue upon 15 minutes of exposure to N-BPs and PMA as a positive control.
Phosphoflow analysis showed consistent induction of c-Jun phosphorylation mediated by Zol, and to a lesser degree by PAM and ALN ( Figure 2F). This increase in phosphorylation by N-BPs was not observed in p65, the active subunit of NFB ( Figure 2G).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

In vivo study population
To examine the effects of N-BPs in vivo, we obtained retrospective PBMCs and plasma samples (baseline, and weeks 2, 24 and 48 post-intervention) from the clinical trial ACTG A5163 that previously assessed the efficacy of ALN vs. placebo to increase bone mineral density in PLWH on ART (36). Eligibility criteria for the ACTG A5163 trial included two consecutive measures of viral suppression (≤5,000 RNA copies by any quantitative HIV viral load assay) prior to enrollment. Our criteria to obtain banked samples were viral suppression and availability of stored samples with more than 5 million PBMCs at study entry, and at least one follow-up sample collection. Plasma samples were used to confirm viral suppression at study entry and at follow-up time points. We received samples from 57 participant and thawed PBMCs demonstrated acceptable viability, integrity, cell numbers and viral suppression (< 40copies/mL) in 44 of the 57 samples, 23 from the ALN group and 21 from the placebo group (Supplementary Table 4).
Samples were randomly assigned to perform the intact proviral DNA assay (IPDA) in nine participants from the ALN group and seven from the placebo group (Supplementary Table 5).
Quantification of HIV-1 caRNA, mass cytometry, and HIV-specific T cell responses by ELISPOT were performed in 15 ALN-treated participants and 14 from the placebo group (Supplementary Table 6). All experiments were assayed blinded.

ALN treatment reactivates the latent HIV-1 reservoir in PLWH
Unspliced HIV-1 gag caRNA levels were measured in 14 participants, nine individuals treated with ALN and five who received placebo. In each individual, between six to 15 replicates of 1x10 6 PBMCs were assayed. Baseline levels were comparable between the two groups ( Figure 3A) and were not associated with baseline CD4 or CD8 T cell counts, time on ART or age (p>0.05 for all . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint comparisons). Seven participants (five treated with ALN) were women and had lower baseline caRNA levels than men ( Figure 3B). Mean longitudinal caRNA levels remained comparable between the two interventions ( Figure 3C) and were lower in women compared to men ( Figure   3D). However, the lack of difference between ALN and placebo groups was due to variable HIV-1 caRNA dynamics in the ALN group, as observed when analyzing individual donors. In eight of nine participants, we observed differential effects of ALN treatment on HIV-1 caRNA ( Figure 3E).  Fig. 4). Given the complex dynamics of HIV-1 caRNA (37)(38)(39), we may be capturing initial latency reversal after which there may be clearance and decay, therefore results may be reflecting different timing and duration of the measurement.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023.

Effect of ALN on the viral reservoir in PLWH
The intact proviral DNA assay ( Table 8). A trend towards a decay from baseline to week two in intact provirus was observed in the ALN group which was not observed in the placebo group (p=0.078 vs. p=1.0, Figure 4D). However, as three individuals from the placebo group had a similar proportional decay in intact proviral DNA levels compared to the three participants with largest decays in the ALN group, a larger cohort is required to confirm this preliminary trend. Similarly, fold changes from baseline to week two after intervention were similar between ALN and placebo groups for all HIV DNA species ( Figure 4E). Finally, we observed differences in changes from baseline to week two in total and defective HIV-1 DNA only in the ALN group ( Figure 4F). Comparable HIV-1 DNA copies over time in both groups ( Figure 4C), the small and uneven sample size (n=9 for the ALN . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Effect of ALN on immune cells
We performed mass cytometry in eight participants from the ALN group and six from the placebo group. Over the course of ALN treatment frequencies of V2 and V1 T cells remained stable whereas effector memory (EM)-CD8 T cell frequencies decreased. ( Figure 5A). All the other cell populations analyzed showed comparable baseline and longitudinal frequencies (Supplementary Figs. 6 and 7). Higher granzyme B (GzmB) production from V2 T cells, V1 T cells and CD8 T cells compared to the placebo group after 24 weeks of intervention were detected ( Figure 5B).
Comparison by biological sex revealed higher baseline GzmB production from V2 T cells, V1 T cells, and CD8 T cells in women than in men, although men experienced a longitudinal increase that reached comparable levels to women ( Figure 5C). Although we did not detect an effect of ALN on HIV-1 specific T cell responses ( Supplementary Fig. 8), there was evidence for changes in V2 T cell functionality ( Figure 5B) that led to an induction of CD8 T cell functions including decreased peripheral EM-CD8 T cells ( Figure 5A) and increased apoptotic GzmB granule production ( Figure 5B).

Increased Granzyme B production associates with caRNA decay
To understand the dynamics of HIV-1 caRNA observed in the ALN group ( Figure 3E) we compared GzmB, IFN- and TNF-α production from three individuals with an induction of HIV-1 caRNA copies, and three with a decay in HIV-1 caRNA levels. Most noticeable changes included a consistent longitudinal increase in GzmB production ( Figure 5D), and calculated percent changes over time showed that V2 T cells, V1 T cells, and CD8 T cells contributed to increasing GzmB production ( Figure 5E). In addition, in participants with a decay in HIV-1 caRNA levels we observed constant V2 T cell frequencies over time, despite being lower than in participants with increased HIV-1 caRNA levels ( Figure 5F), lower frequency of naïve CD8 T cells and higher . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint 14 frequency of EM-CD8 T cells ( Figure 5G). Other comparisons are shown in Supplementary Fig.   9. These immune differences may contribute to the variable HIV-1 caRNA slopes and warrants further investigation in a larger cohort.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Donors
For ex vivo studies, frozen samples from PLWH on ART with undetectable plasma viral load (<50 copies/mL) for at least one consecutive year before inclusion in this study were obtained at the University of North Carolina, USA. Participants' characteristics have been previously reported (36). Frozen peripheral blood mononuclear cells (PBMCs) and plasma samples from the AIDS Clinical Trial Groups (ACTG) A5163 clinical trial performed in 2004 were requested for in vivo analyses (36). The only criteria to obtain the samples was that participants had to be virally suppressed and have cell availability of at least 5 million in the baseline and one follow-up timepoint. Briefly, the objective of that study was to assess the effect of once-weekly oral administration of ALN on bone mineral density. A5163 was a phase II, double blind, placebocontrolled study where chronic ART-suppressed HIV-1-seropositive participants were randomized into ALN and placebo interventions. Both arms took calcium carbonate and vitamin D and were followed for 48 weeks. PBMCs and plasma samples were obtained and stored prior to the intervention (basal) and at weeks 2, 24, and 48 post-intervention. All assays were performed blinded as per treatment status.

Drugs and Compounds
PAM, Zol and ALN were used at concentrations that mimic plasma concentrations achieved in the blood with FDA-approved doses (28,29). PAM was used at 2.5g/mL or 25g/mL, Zol at 1g/mL, and ALN at (2.5µM). In some experiments, we treated cells in parallel with 350nM VOR (for HIV-1 caRNA levels quantification), or 500nM (for replication-competent HIV-1 determination). PAM at 2.5g/mL and 25g/mL was able to similarly induce HIV-1 caRNA and replication competent HIV-1-1, and was compared to VOR in some experiments.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Quantitative Polymerase Chain Reaction (qPCR)
For ex vivo analysis of HIV-1 caRNA levels, magnetically isolated rCD4T cells from PLWH on suppressive ART were exposed to N-BPs, PHA and IL-2, or left untreated. In some experiments, cells were exposed VOR. After six hours of exposure, rCD4 T cells were washed and plated in 6-15 replicates of 1x10 6 cells per condition, depending on cell availability. After further washes, cells were pelleted and stored at -80 o C, until RNA isolation. For the in vivo part of the study, thawed PBMCs were rested overnight, plated in 3-5 replicates of 1x10 6 cells (depending on cell availability), and stored at -80 o C. Automated RNA extraction was performed (KingFisher, ThermoFisher Scientific), cDNA was synthesized using standardized protocols, and qPCR performed in triplicate using specific and validated primers, probes, and standard curves to amplify unspliced HIV-1 gag, as previously described (40). Results were expressed as HIV-1 gag copies per 10 5 rCD4 T cells for the ex vivo analysis and 10 5 PBMCs for the in vivo analysis. The limit of detection of the PCR was 10 HIV-1 gag RNA copies/10 5 cells.

RNA-Sequencing
In order to explore the mechanism of action of N-BPs related to HIV latency reactivation, RNA was isolated from rCD4 T cells exposed to PAM or VOR for 6 hours, or left untreated.
RNA isolation and quality control. 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).

Intact Proviral DNA Assay (IPDA)
Total DNA was extracted from frozen cell pellets of 0.3-1x10 6 PBMCs (Qiagen, Germantown, MD), and concentrated using an ethanol precipitation protocol with GlycoBlue following manufacturer's instructions (Thermofisher, Frederick, MD). DNA concentration and integrity were measured by Nanodrop spectrophotometer (Thermofisher). Intact, hypermutated and/or 3' defective, and 5' defective HIV-1 copies/million cells were determined by droplet digital PCR . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (45), where HIV-1 and human RPP30 reactions were conducted independently in parallel, and copies were normalized to the quantity of input DNA. Briefly, primers and probes have been previously reported (46), and, in each ddPCR reaction, a median

Mass Cytometry
Mass cytometry was performed in eight individuals treated with ALN and seven who took placebo using an optimized panel of 32 markers.

Panel design and conjugation of antibodies. Pairing of metals with antibodies was conducted using
Fluidigm software (San Francisco, CA). When commercial antibodies were not available, custom conjugations were performed using MaxPar X8 Antibody Labeling Kit (Fluidigm). Cell populations were phenotypically defined as previously reported (47,48) and described in Supplementary Table S9.
Sample Preparation. In each experiment, 3x10 6 PBMCs (pre-stained with CD45/89Y) were combined with 5x10 5 spiked control PBMCs from a single HIV-1-infected control donor that was consistently used in all the experiments (pre-stained with CD45/115In), as described (49). Prior to 30min RT incubation with surface marker antibodies in Cell Staining Buffer (CSB, Fluidigm), cell . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. Single cell data analysis. Data files were bead normalized and debarcoded using software provided by Fluidigm. Analysis was performed in collaboration with Astrolabe Diagnostics (50). Singlecell data was clustered using the FlowSOM R package (50,51). Differential abundance analysis was done using the edgeR package (52, 53) following a previously reported method (54). Data is presented as relative frequency of cell populations related to the total number of alive events acquired. Cluster labeling, method implementation, and visualization were done through the Astrolabe Cytometry Platform (Astrolabe Diagnostics, Inc.).

HIV-1-specific T cell responses
ELISPOT was performed as previously reported (55). Briefly, cryopreserved PBMCs from participants treated in vivo with ALN or PLACEBO were thawed and rested overnight then added . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made to ELISpot plates (Merck, Millipore) in quadruplicate (1 or 1.25 × 10 5 PBMC/ well) with peptides pools of optimal CD8 T cell epitopes from either HIV-1-1 Gag/Nef (n=109, 2µg/ml, CTL-A) or Influenza, HCMV, and Epstein-Barr Viruses (n= 55, 2µg/ml, FEC55). PHA positive controls and media-only controls were also run. Samples were counted independently three times, and cell counts were averaged resulting in <10% variation. No minimum cell viability or cell recovery criteria was applied. Timepoints for each participant were run together. Antigens in thawed peptide plates were mixed with 1:1 with PBMCs in the ELISpot plate to a final concentration of 2 μg/ml and incubated for 18-20 hours at 37°C, 5% CO2. Coating, development (MabTech), and reading of ELISpot plates (AID Reader) has been described previously. Positive T cell responses were defined as ≥30 SFU per million, > 4 times the average of replicate background wells. Zero values were not accepted in any replicate of antigen-stimulated wells.

Statistics
Nonparametric, two-sided, exact tests were used to make comparisons. A Mann-Whitney U test was used for comparisons between different groups, a Wilcoxon signed-rank test was used for analyzing repeated measures within the same groups, and a Fisher's exact test was used for comparisons between categorical variables. Values for ex vivo analysis of HIV-1 gag caRNA below the limit of quantification (LOQ) were assigned a value of half the limit of quantification (LOQ=5 copies/mL). SLDAssay,(56) a software package in R for serial limiting dilution assays, was used to compute maximum-likelihood bias-corrected estimates for IUPM, accompanied by an exact 95% CI and p-value for goodness of fit (PGOF). Tests for trend with increasing time on intervention were performed using a two-sided

Study Approval
All participants provided written informed consent prior to inclusion in the study, and studies were approved by the George Washington and University of North Carolina Institutional Review Board.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint

DISCUSSION
In this work, we show the novel use of N-BPs as LRAs. Based on a careful examination of HIV-1 caRNA and replication-competent virus production after ex vivo exposure of isolated rCD4 T cells from PLWH on ART, we demonstrate the capacity of N-BPs to reverse latency. These results were further corroborated using samples from in vivo treated PLWH from a previous clinical trial (36).
We detected perturbations of the viral reservoir consisting of an induction in HIV-1 caRNA in four participants, and a decay in three others in whom a decay in total HIV-1 DNA was also observed.
However, limited sample size and cell availability precluded providing evidence of an impact on the viral reservoir. Despite these limitations, we show that N-BPs exert a potent induction of viral replication, a finding that warrants further clinical investigation.
HIV-1 caRNA reflects the size of the HIV-1 reservoir (38,57) since it represents intracellular transcripts that originate from latently infected cells in virally suppressed PLWH (37,38). Confirming previous studies (58,59), women on ART had lower frequency of infection measured by HIV-1 caRNA. ALN induced perturbations on HIV-1 caRNA in 78% of the participants which included both increased and decreased levels upon intervention, highlighting the complexity of caRNA dynamics. Variability of the HIV-1 caRNA slopes in our study may reflect when and for how long the LRA effect is measurable after once a week ALN administration.
Encouragingly, in three of the ALN-treated participants changes in HIV-1 caRNA coincided with a decay in total HIV-1 DNA levels. Similarly, a recent study also observed variable HIV-1 caRNA slopes with increases and decreases after treatment with a broadly neutralizing antibody and VOR, although no changes in DNA were detected (39).
Previous studies have shown decay for intact but not defective HIV-1 DNA species in PLWH on ART (60)(61)(62). Due to sample limitations, our study used total PBMCs for IPDA. We . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint 24 found that levels of intact provirus measured in PBMC were similar to previous reports and correlated with total HIV-DNA, as previously reported (46,(59)(60)(61)(63)(64)(65). Participants in our small study cohort had received ART for a mean of 4.4 years and changes in intact, total or defective HIV-1 species did not associate with time of exposure to ART. Overall, levels of HIV-1 DNA species were comparable between ALN-treated and placebo groups throughout the study. We did however detect a trend towards a decay from baseline to week two in the ALN group in intact provirus. By contrast, we found no evidence of decay either in total and defective HIV-1 DNA in the placebo group. Overall, while our results are encouraging, they need to be interpreted cautiously due to our small sample size, the inherent variability of the assay and complex reservoir dynamics, especially during the initial years of ART exposure. Therefore, the potential effect of ALN on the viral reservoir warrants further investigation using a larger cohort with the specific objective of analyzing the novel use of ALN as an LRA and immunomodulatory agent. We propose a model whereby N-BPs induce both HIV-1 reactivation and direct activation of V2 T cells' effector functions, that in turn enhance cytotoxic properties from other effector cells (20,21,68,69). For the reactivation effect, although further investigation is required to understand how N-BPs reactivate latent HIV, our results suggest that c-Jun, but not NFB signaling, leads to viral reactivation. Increased phosphorylation at the Ser73 residue of c-Jun (35) after exposure to Zol, provides evidence of the involvement of this pathway on viral reactivation (33,34). In our experiments, cells were exposed to one single N-BPs dose for 15 minutes and only one phosphorylation site was analyzed. In addition, since Zol has been reported to be the most potent N-BP (70), in vitro exposure to PAM or ALN may require higher doses or longer times to detect an effect in c-Jun phosphorylation, and possibly other residues and/or AP-1 components.
One of the advantages of investigating FDA-approved drugs for use in other indications includes the known safety and toxicity profiles. ALN's safety profile is well known and, although there are side effects that need to be carefully monitored, these drugs are currently used as firstline treatment for osteoporosis (31). We have discovered that N-BPs are a novel class of potent LRAs that induce both reactivation of persistent HIV-1 and effector cellular functions in PLWH on suppressive ART. We present the first evidence in humans that treatment with N-BPs may be further pursued in the context of HIV cure, possibly in combination with other interventions aimed at enhancing pre-existing immunity.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023.

DECLARATION OF INTERESTS
The Authors declare no competing interests

ADDITIONAL INFORMATION
Supplementary Information is available for this paper Further information and requests for resources and reagents should be directed to and will be fulfilled by Natalia Soriano-Sarabia.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint

Supplementary Material
Supplementary material includes: Figures S1-S9 Supplementary Tables S1-S9: Table S1: Characteristics of PLWH included in ex vivo experiments Table S2: RNA-seq data: differentially expressed genes (will be uploaded to a public database) Table S3: GSVA pathway analysis (will be uploaded to a public database)      . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  T cell  CD3+ CD19-CD33-, TCRvd1-TCRvd2+  V1 T cell  CD3+ CD19-CD33-, TCRvd1+ TCRvd2-CD8+ T cells (naïve)  CD3+ CD19-CD33-, TCRvd1-TCRvd2-, CD4-CD8+ CD56-, CD45RA+,  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint Figure S1: HIV-1 caRNA levels in 23 PLWH on suppressive ART. Individual plots show HIV-1 gag caRNA copies/mL in the different conditions tested in each experiment depending on cell availability. A) Comparison of PAM, Zol and ALN with PHA. B) In Pt12 through Pt18, the capacity of N-BPs to reactivate latent HIV was compared to 500nM VOR. Each graph represents one participant, and each symbol represents one biological replicate of 1x10 6 isolated rCD4 T cells. Comparison of HIV gag copies induction between C) PAM at 2.5µg/mL and 25µg/mL) and D) PAM at 25µg/mL and VOR. Pt, participant. P-values were calculated by a Wilcoxon signed-rank test, and only p-values < 0.05 are displayed. d Figure S2: Ex vivo viability upon treatment with PAM and Zol. PBMCs from PLWH on suppressed ART were exposed to 2 g/mL PHA+100 U/mL IL-2 or different concentrations of A) PAM or B) Zol as specified, and viability (7-AAD) measured by flow cytometry in total CD4 T cells, CD8 T cells,  T cells, and resting CD4 (rCD4) T cells. The means of repeated experiments in three to six individuals are presented.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint Figure S9: Comparison of IFN- and TNF-α production from different effector cell populations (N=8) in A) ALN or placebo groups, B) women and men treated with ALN. C) Comparison of frequencies of circulating cell populations according to the HIV caRNA slope (N=3 negative trend, grey, and N=3 positive trend, blue). Mean ±SEM is presented. Mann-Whitney U test.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.07.527421 doi: bioRxiv preprint