Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo

Abstract

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2,3,4,5,6,7,8,9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow–liver–thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal—in combination with appropriate tools for systemic clearance of persistent HIV infection—greatly increases opportunities for HIV eradication.

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Fig. 1: Efficient in vitro AZD5582 target engagement and induction of HIV transcription.
Fig. 2: AZD5582 induces HIV RNA expression in resting CD4+ T cells from tissues of HIV-infected ART-suppressed BLT mice.
Fig. 3: AZD5582 induces SIV RNA expression in the plasma and lymph nodes of ART-suppressed SIV-infected rhesus macaques.
Fig. 4: AZD5582 specifically activates the ncNF-κB pathway in SIV-infected ART-suppressed rhesus macaques without generalized T cell activation.

Data availability

Source Data for Figs. 14, Extended Data Figs. 1, 2, 4, 610 and Supplementary Tables 46 are provided with the paper. Gene-expression data are available at the Gene Expression Omnibus (GEO) repository (accession number GSE141546 and GSE142774). Any other data are available from corresponding authors on reasonable request.

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Acknowledgements

We thank Garcia and Chahroudi laboratory members, the Animal Histopathology & Laboratory Medicine Core at the University of North Carolina-Chapel Hill (UNC-CH), which is supported in part by an NCI Center Core Support Grant (5P30CA016086-41) to the UNC Lineberger Comprehensive Cancer Center and technicians from the Department of Comparative Medicine at UNC-CH; the HIV/STD Laboratory Core and the Clinical Pharmacology and Analytical Chemistry Core of the UNC Center for AIDS Research (CFAR) (P30 AI050410); D. Hazuda, B. Howell and S. Barrett (Merck & Co.) for assistance with ART-containing chow; Yerkes Animal and Research Resources; the Children’s Healthcare of Atlanta and Emory University Pediatric Flow Cytometry Core, Emory CFAR Translational Virology and Reservoir Cores, the Quantitative Molecular Diagnostics Core of the AIDS and Cancer Virus Program, Frederick National Laboratory, as well as GSK for tenofovir disoproxil fumarate, emtricitabine and dolutegravir. This work was supported by the National Institutes of Allergy and Infectious Diseases (NIAID)(AI123010, AI096113, AI111899, AI117851 and P30 AI050410), and Mental Health (NIMH) (MH108179). This work was supported by the Emory Consortium for Innovative AIDS Research in Nonhuman Primates (UM1 AI124436), amfAR (109353-59-RGRL), the Yerkes National Primate Research Center (P51 OD011132) and the Translational Virology and Reservoir Cores of the Center for AIDS Research at Emory University (P30 AI050409). Research was also supported by Qura Therapeutics and by CARE, a Martin Delaney Collaboratory (1UM1AI126619-01) of the NIAID, NINDS, NIDA and NIMH. By the Natural Science Foundation of Guangdong Province, China (2016A030310108), UNC-South China STD Research Training Center (1D43TW009532) and Chinese National Key Technologies R&D Program for the 13th Five-year Plan (2017ZX10202101003). Federal funds were used for this research from the National Cancer Institute, NIH (contracts HHSN261200800001E and 75N91019D0002). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

Author information

J.V.G., A.C., G.S., R.M.D., A.W. and D.M.M. conceived and designed the studies. G.C.S., D.M.I., S.R.W., S.D.F., C.G., E.P.B. and R.G.F. assessed in vitro AZD5582 activity. M.K., Z.W., J.H.B., D.F. and C.D.J. performed the in vitro transcriptomic study. D.F. and J.-P.R. contributed clinical specimens. C.C.N., P.T.H., C.D., N.J.S. and B.L. performed mouse experiments. R.A.S. and C.G.C. performed the qPCR analysis of HIV RNA levels in mouse samples. R.A.C. performed the immunohistochemical analysis. N.M.A. isolated human resting CD4+ T cells. C.C.N. analysed mouse data. M.M., A.D.B. and S.J. performed the monkey experiments. G.C.S., J.D., S.M. and R.M.D. conducted pharmacokinetic studies in NHP. C.M. and N.S. isolated resting CD4+ T cells from macaques. T.H.V. supervised the qPCR analyses of SIV RNA and DNA levels in monkey samples. M.M. and A.D.B. performed flow cytometry and analysed monkey data. G.K.T., A.A.U. and H.W. performed the macaque RNA-seq analyses and S.E.B. analysed these data. C.M.F. and B.F.K. performed env sequencing analyses. J.D.L. supervised the ultrasensitive plasma viral loads. G.C.S. and C.G. analysed AZD5582 target engagement in human, mouse and macaque samples and J.D. and R.M.D. analysed the data. C.G. performed the ELISPOTs. C.C.N., M.M., A.W., A.C. and J.V.G. wrote the manuscript with input from all authors.

Correspondence to Richard M. Dunham or Ann Chahroudi or J. Victor Garcia.

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Peer review information Nature thanks Mathias Lichterfeld and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Short-duration exposure to AZD5582 activates the ncNF-κB pathway.

ad, Isolated total human CD4+ T cells treated with 100 nM AZD5582 and then either not washed (a) or washed three times with PBS 8 h (b), 4 h (c) or 1 h (d) after treatment. Whole-cell lysates were then analysed by immunoblot for components of the ncNF-κB pathway. e, Densitometry analysis of the ratio of p52 to p100 from the pulse-wash assay immunoblots. Points represent values for the densitometric ratio from one western blot, representative of several independent experiments. ae, Entire set representative of 1 experiment with 5 replicates of the 1-h wash condition conducted. f, Fold induction of ncNF-κB target gene expression in isolated CD4+ T cells from an uninfected donor treated with the indicated concentrations of AZD5582 and either washed after 1 h or not washed, and subsequently cultured for 24 h as measured by RT–qPCR. Points represent two technical replicates and lines represent the mean. The data presented are representative of three independent experiments. g, DMSO-normalized induction of luciferase activity from the Jurkat reporter model after exposure to AZD5582 (10, 100 or 1,000 nM) for 30 min (blue), 1 h (red), 2 h (green) or continued exposure (purple). Points represent three replicates in one assay run, representative of two independent experiments. Lines represent the mean of the three replicates. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 2 AZD5582 induces HIV RNA expression in resting CD4+ T cells from tissues of HIV-infected, ART-suppressed BLT mice.

HIV RNA levels in resting CD4+ T cells isolated from the bone marrow, lymph nodes, liver, lung and spleen of control (Cntrl, blue circles) or AZD5582-treated (AZD, red circles) mice (cells pooled from n = 6 mice per group for each tissue) were analysed in triplicate. Statistical significance was determined with a two-sided Student’s t-test. The mean fold increase in viral RNA levels in resting CD4+ T cells from tissues in the experiment shown in Fig. 2 was 12.1 (±3.7), whereas in this figure it was 8.4 (±6.0). These values were not statistically different (P = 0.4286, two-sided Mann–Whitney U-test). Data are mean ± s.e.m. Source data

Extended Data Fig. 3 AZD5582 ex vivo target engagement.

a, Western blot analysis of p100, p52 and cIAP1 protein levels in cells isolated from the bone marrow, lymph node and spleen of BLT mice before and 20 h after ex vivo treatment with AZD5582. Loading control, β-actin. Representative of two experiments. b, cIAP expression in resting CD4+ T cells isolated from the thymus, spleen, lymph nodes, liver, lung and bone marrow. Loading control, β-actin. Representative of two experiments. c, cIAP expression in the thymic organoid of HIV-infected ART-suppressed BLT mice 48 h after the administration of vehicle control or AZD5582 (three control and one AZD5582-treated analysed). Positive cells, brown. Imaged at 4× and 40× magnifications; scale bars, 100 μm (4×) and 50 μm (40×). Boxes in the 4× images indicate regions corresponding to images at 40× magnification. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4 Pharmacokinetic and pharmacodynamic assessment of AZD5582 in rhesus macaques.

a, AZD5582 (0.1 mg kg−1) was administered to healthy rhesus macaques (n = 3) by intravenous infusion. Plasma concentrations of AZD5582 (left y axis) are shown for the indicated time points. Flow cytometry was used to measure intracellular p100 levels, shown as the geometric mean fluorescence intensity (gMFI) in CD4+ T cells and plotted as the percentage of baseline of p100 gMFI (right y axis). b, Plasma concentrations of AZD5582 after one (dark red), three (red), six (pink) or ten (orange) doses in six SIV-infected ART-treated rhesus macaques (RM) and after one dose in three uninfected control rhesus macaques (grey). Individual values are shown as symbols. c, Western blot analyses of inactive p100 and active p52 forms of NF-κB2 in lymph-node mononuclear cells collected 48 h after the third or tenth dose of AZD5582 in SIV-infected ART-suppressed rhesus macaques (red; n = 3 for both the 3-dose and 10-dose groups) or at equivalent time points for placebo controls (blue; n = 2 for both the 3-dose and 10-dose groups). Immunoblots are shown in the top panels and densitometry analyses of the p52:p100 ratios are shown in the bottom panels. The line represents the median. d, Cryopreserved control rhesus macaque splenocytes were treated with the indicated concentrations of AZD5582 for 48 h, then p100/p52 levels were analysed by western blotting to measure engagement of the ncNF-κB pathway. e, DMSO-normalized densitometric p52:p100 ratio versus the AZD5582 concentration. For d, e, the experiments were performed in duplicate. f, Cryopreserved rhesus macaque splenocytes were exposed to DMSO alone (Untx), 100 nM AZD5582 washed off after 1 h and cultured for 47 h (Pulsed) or continuous 100 nM AZD5582 for 48 h (Cont.), after which the cells were studied by western blot for p100 and p52 levels. g, Densitometric p52:p100 ratio. For f, g, data represent a single experiment. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 5 Phylogenetic trees based on full env sequencing.

Plasma virus was sequenced from four time points per macaque (n = 5): near peak viraemia (2 weeks after infection; red), immediately before ART (8 weeks after infection; orange) and two time points of on-ART viraemia during AZD5582 treatment (green and blue). All sequences per macaque were phylogenetically analysed and the resulting phylogenetic trees are shown for each macaque. The horizontal bar indicates the genetic distance. nt, nucleotide.

Extended Data Fig. 6 SIV DNA levels in total CD4+ T cells and replication-competent reservoir size in ART-suppressed SIV-infected rhesus macaques.

a, Longitudinal assessment of cell-associated SIV DNA levels in total CD4+ T cells isolated from peripheral blood and lymph nodes of AZD5582-treated (n = 12, red) and control (n = 4, blue) ART-suppressed SIV-infected rhesus macaques. Grey shading represents the period of ART administration. b, Comparison of cell-associated SIV DNA levels in total CD4+ T cells isolated from the lymph nodes and peripheral blood of AZD5582-treated and control ART-suppressed SIV-infected rhesus macaques. Total CD4+ T cells were analysed from AZD5582-treated rhesus macaques 48 h after receiving 3 doses (lymph nodes and peripheral blood, n = 3) or 10 doses (lymph nodes and peripheral blood, n = 9) of AZD5582. Total CD4+ T cells were analysed from placebo-control rhesus macaques (lymph nodes and peripheral blood, n = 4) at equivalent time points. Open symbols indicate AZD5582-treated rhesus macaques with on-ART viraemia above 60 copies per ml of plasma. Statistical significance was determined using a two-sided Mann–Whitney U-test. c, Quantitative viral outgrowth assays were performed for AZD5582-treated rhesus macaques 48 h after receiving 3 doses (lymph nodes, n = 2; spleen, n = 3) or 10 doses (lymph nodes, n = 3; spleen, n = 2) of AZD5582. Quantitative viral outgrowth assays were performed from control rhesus macaques (lymph nodes and spleen, n = 4) at equivalent time points. Open symbols indicate AZD5582-treated rhesus macaques with on-ART viraemia. Statistical significance was determined with a two-sided Mann–Whitney U-test. Horizontal lines represent the median (b, c). Source data

Extended Data Fig. 7 AZD5582-induced gene pathways and genes in SIV-infected ART-suppressed rhesus macaques.

a, DAVID analysis showing pathways significantly enriched in genes differentially expressed after AZD5582 treatment relative to baseline in CD4+ T cells isolated from lymph nodes (black bars) and peripheral blood (grey bars). n = 6 for both lymph nodes and peripheral blood; for each, n = 3 for 3 doses of AZD5582 and n = 3 for 10 doses of AZD5582. b, Leading edge genes from the ‘hallmark TNF signalling via NF-κB’ pathway from MSigDB. Genes were identified in the leading edge of CD4+ T cell samples from the lymph nodes before and after treatment with AZD5582 (shown in Fig. 4b). The contrast depicted is the fold change of each gene for each rhesus macaque’s post-treatment sample relative to the pre-treatment values for CD4+ T cells from the lymph nodes (top) and the peripheral blood (bottom). n = 6 for both lymph nodes and peripheral blood; for each, n = 3 for 3 doses of AZD5582 and n = 3 for 10 doses of AZD5582. c, Heat map of cNF-κB (top) and ncNF-κB (bottom) pathway gene expression in rhesus macaques with (left, n = 5) or without (right, n = 6) on-ART viraemia of >60 copies per ml plasma. One rhesus macaque without on-ART viraemia was excluded from this analysis because of technical issues (higher than expected unmapped and multi-mapped reads, and lower than expected unique identified reads compared to the means). Colour scale, log2-transformed fold changes of post-treatment compared with pre-treatment values. Source data

Extended Data Fig. 8 Relationship between virological status before intervention and virological response to AZD5582 treatment in SIV-infected ART-suppressed rhesus macaques.

a, Individual representations of plasma SIV RNA levels measured by ultrasensitive assay (limit of detection 3 copies per ml of plasma) before and during AZD5582 treatment in SIV-infected ART-suppressed rhesus macaques (n = 12). ‘Baseline’ shows two or three plasma viral loads before AZD5582 treatment and ‘AZD5582’ shows three or four plasma viral loads during AZD5582 treatment. Open symbols indicate the five AZD5582-treated rhesus macaques with on-ART viraemia of >60 copies of SIV RNA per ml of plasma using the standard viral load assay (confirmed with ultrasensitive assay). The orange box highlights an additional 3 rhesus macaques who did not experience on-ART viraemia of >60 copies per ml of plasma but had ≥2 SIV RNA measurements above baseline values using the ultrasensitive quantification method. b, Comparison of the medians of plasma SIV RNA levels at baseline and during AZD5582 treatment measured by ultrasensitive assay for ART-suppressed SIV-infected rhesus macaques (n = 12). Statistical significance was determined with a Wilcoxon matched-pairs signed-rank test. c, Comparison of the levels of plasma SIV RNA at peak, plasma SIV RNA before ART initiation, SIV DNA in peripheral-blood CD4+ T cells before AZD5582 treatment (pre-LRA), SIV DNA in lymph-node CD4+ T cells pre-LRA, SIV RNA in peripheral blood CD4+ T cells pre-LRA and SIV RNA in lymph-node CD4+ T cells pre-LRA in rhesus macaques with increased plasma viral loads (PVL) by standard and/or ultrasensitive assay during AZD5582 treatment (increased plasma viral loads, n = 8) compared with rhesus macaques that did not demonstrate increased viral loads (stable plasma viral loads, n = 4). Statistical significance was determined using a two-sided Mann–Whitney U-test. Horizontal lines represent the median (a, c). Source data

Extended Data Fig. 9 AZD5582 can be safely administered in SIV-infected ART-suppressed rhesus macaques.

ac, Longitudinal assessment of serum chemistries (a), complete blood counts (b) and weight (c) of SIV-infected ART-treated rhesus macaques treated with AZD5582 (red, n = 12) compared with controls (blue, n = 9). Grey shading represents the period of ART administration. AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; GGT, γ-glutamyltransferase; HGB, haemoglobin; WBC, white blood cell. Source data

Extended Data Fig. 10 Immunological effect of AZD5582 on SIV-infected ART-suppressed rhesus macaques.

a, b, Longitudinal assessment of the count (a) and the frequency and viability (b) of CD4+ T cells in SIV-infected ART-treated rhesus macaques during the period of AZD5582 treatment (n = 12). Lines represent median values. c, d, CD4+ T cell naive and memory subset frequencies (c) and their expression of Ki-67 (d) in SIV-infected ART-treated rhesus macaques during the period of AZD5582 treatment (n = 12). Lines represent median values. e, SIV gag-specific (left) and SIV env-specific (right) CD8+IFNγ+ T cell responses in SIV-infected, ART-suppressed, AZD5582-treated (red, n = 6) and control (blue, n = 4) rhesus macaques. SFU, spot-forming units. f, Pie charts depicting the ability of memory CD8+ T cells isolated from SIV-infected ART-suppressed control rhesus macaques (n = 5) to produce IFNγ, IL-2 and/or TNF in response to stimulation with PMA and ionomycin in the absence (left) or presence (right) of AZD5582 pre-treatment. g, Memory CD8+ T cell proliferative response to stimulation with PMA and ionomycin with or without AZD5582 pre-treatment. Top, representative flow cytometry dot plots gated on memory CD8+ T cells. Bottom, comparison between divided cells 5 days after stimulation in each group (n = 5 for each). Statistical significance was determined with a Wilcoxon matched-pairs signed-rank test. Horizontal lines represent the median. h, Longitudinal assessment of plasma levels of IP-10, MIP-1β, MCP-1, IL-6 and IL-10 by multiplex assay in SIV-infected ART-suppressed rhesus macaques treated with AZD5582 (red, n = 6) or control rhesus macaques (blue, n = 4). An additional four analytes (IFNγ, IL-8, IL-1β and IL-2) were undetectable in all macaques. ae, h, Grey shading represents the period of ART administration. Dashed lines represent AZD5582 or placebo infusions. LLOD, lower limit of detection. Source data

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-2 and Supplementary Tables 1-7.

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Supplementary Table 8

Gene expression in AZD5582-treated on-ART viremic and on-ART non-viremic RM.

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Nixon, C.C., Mavigner, M., Sampey, G.C. et al. Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo. Nature 578, 160–165 (2020). https://doi.org/10.1038/s41586-020-1951-3

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