Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells

Abstract

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1,2,3,4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection.

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Fig. 1: Study design and phenotypic and transcriptomic effects of N-803 with or without CD8 depletion in rhesus macaques.
Fig. 2: SIV and HIV reactivation after CD8 depletion combined with N-803 treatment.
Fig. 3: In vitro co-culture of latently infected human CD4+ T cells with autologous CD8+ T cells results in decreased expression of HIV Gag during LRA administration.
Fig. 4: CD8 depletion combined with N-803 administration does not decrease the size of the latent SIV viral reservoir.

Data availability

Illumina sequencing reads for RNA-sequencing experiments were submitted to the NCBI SRA repository (accession number SRP188630). RNA-sequencing datasets were submitted to the NCBI GEO repository (accession number GSE128415). env nucleotide sequences have been deposited in the GenBank (accession numbers MK922999MK923550).

Change history

  • 04 February 2020

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This work was supported by NIH grants R01-AI125064 and UM1-AI124436 (to G.S. and A.C.); R01-AI143414 (to G.S. and D.A.K.); R01-MH108179 and R01-AI111899 (to J.V.G.); UM1-AI126619 (to D.M.M.); R01-AI123010 (to A.W.); P30 AI050409 (Emory Center for AIDS Research); P51 OD011092 (Oregon National Primate Research Center base grant); the National Institutes of Health’s Office of the Director, Office of Research Infrastructure Programs P51OD011132 (Yerkes National Primate Research Center base grant); and supported in part by federal funds from the National Cancer Institute, National Institutes of Health, under contracts HHSN261200800001E and 75N91019D00024 (J.D.L.). We thank B. Jones, S. O’Connor and J. Sacha for discussions; S. Ehnert, S. Jean and all of the animal care and veterinary staff at the Yerkes National Primate Research Center; B. Cervasi and K. Gill at the Emory University Flow Cytometry Core; Emory and Pediatric’s/Winship Flow Cytometry Core; the Translational Virology and Reservoir Cores of the Emory CFAR, the Emory Nonhuman Primate Genomics Core for RNA sequencing and analysis and the Quantitative Molecular Diagnostics Core of the AIDS and Cancer Virus Program, Frederick National Laboratory, for high-sensitivity plasma viral-load testing; NantKwest for providing N-803, K. Reimann and the NHP Reagent Resources for the MT807R1 antibody, R. Geleziunas and Gilead Pharmaceuticals for providing tenofovir and emtricitabine, D. Hazuda and B. Howell from Merck for providing raltegravir and J. Demarest and ViiV Healthcare for providing dolutegravir for this study.

Author information

J.B.M., A.C., M.P. and G.S. designed the experiments. J.B.M., M.M., E.W. and D.G.C. performed the experiments. S.A.S. performed single-genome PCR and sequencing of SIV RNA. S.A.S. and C.A.D. performed the sequence-based analyses and wrote the relevant portions of the manuscript. J.D.L. performed ultrasensitive viral-load analyses. B.C. performed FACS of live cells. T.H.V. measured viral load, and cell-associated DNA and RNA. J.D.E. and K.B.-S. performed RNAscope analysis. S.E.B., G.K.T. and H.W. performed RNA-sequencing analysis. M.K., C.R.A.-S. and W.O.T. constructed BLT humanized mice. M.K. performed the HIV infection and ART suppression of BLT humanized mice. R.A.S. performed the viral load measurements, the isolation of nucleic acids and the analysis of tissue RNA levels for BLT humanized mice. C.R.A.-S. and W.O.T. designed and performed the N-803 and CD8 T cell depletion experiments in BLT humanized mice and analysed the data. A.W. supervised the data collection, analysis, figure preparation and reporting of all samples from the BLT humanized mice. J.V.G. designed, coordinated and supervised all of the BLT experimental work. C.R.A.-S., J.V.G., A.W. and W.O.T. wrote and revised the BLT humanized mice portions of the manuscript. L.F. and D.A.K. performed the in vitro studies and wrote the relevant portions of the manuscript. D.M.M., J.T.S. and J.H.L. provided technical support. J.B.M., A.C. and G.S. wrote the manuscript.

Correspondence to Guido Silvestri.

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Extended data figures and tables

Extended Data Fig. 1 MT807R1 effectively depletes CD8+ T cells in peripheral blood, lymph node and rectum of macaques in addition to depleting NK cells from the blood at day 7.

The percentage of CD8+ cells in the CD3+ population 7 days after depletion was compared to the levels before depletion. ac, Sample flow cytometry shows the absence of CD8β+ cells as part of the CD3+ T cell population after depletion in the peripheral blood (a), rectum (b) and lymph nodes (c) of macaques. Similar results were found across all CD8-depleted macaques (n = 28 biologically independent samples). d, The percentage of CD8β+ cells compared to pre-depletion baseline was calculated in all CD8-depleted macaques (treated with or without N-803, n = 28 macaques) across blood and tissue samples (no differences in CD8+ T cell depletion were observed between groups 2 and 3 on day 7). A two-sided Friedman test was used to calculate statistically significant changes compared to baseline across tissues. e, Depletion of NK cells in the peripheral blood was assessed 1 week after CD8 depletion alone (n = 14 macaques) compared to baseline. Statistical significance was calculated using Wilcoxon signed-rank test. Data are mean ± s.e.m.

Extended Data Fig. 2 Phenotypic changes to CD4+ T cells after intervention.

Longitudinal flow cytometry analysis after treatment with only N-803 (green, n = 7 macaques), only CD8 depletion (blue, n = 14 macaques) and after CD8 depletion and treatment with N-803 (red, n = 14 macaques). a, CD4+ T cell frequency. bf, Percentage of naive (b), stem cell memory (SCM) (c), central memory (CM) (d), transitional memory (TM) (e) and effector memory (EM) (f) CD4+ T cells. gl, Percentage of bulk CD4+ T cells that express PD-1 (g), CD25 (h), CD69 (i), HLA-DR (j), CCR5 (k) and both CCR5 and Ki-67 (l). mv, CCR5 (mq) and Ki-67 (rv) expression in different subsets of CD4+ T cells. Data are mean ± s.e.m. Two-sided Kruskal–Wallis tests were used to compare values after the intervention to the pre-intervention baseline and approximate P-value summaries are provided.

Extended Data Fig. 3 SIV-associated genes and IL-15 subunit genes show a transient change in expression after treatment with N-803 alone.

RNA was extracted from sorted peripheral bulk CD4+ T cells (CD3+CD4+CD8CD20CD14) (left), bulk CD8+ T cells (CD3+CD4CD8+CD20CD14) (middle) and NK cells (CD3CD20CD14NKG2A+) (right) and libraries were prepared, normalized, pooled and clustered on flow cells for sequencing. RNA-sequencing data were aligned to the MacaM v.7.8 assembly of the Indian rhesus macaque genome. Transcripts were analysed for alignment against a custom gene set with SIV host restriction factors, PPIA (capsid folding protein), SIV receptors, SIV receptor agonists, NF-κB subunits (involved in mediating the transcription of long-terminal repeat regions), IL-15 receptor subunits and NFAT subunits. D, day; W, week.

Extended Data Fig. 4 Quantification of levels of cell-associated SIV RNA in peripheral CD4+ T cells before and after interventions.

ac, Changes in expression of SIV RNA in relation to the number of copies of CD4 after intervention with N-803 alone (n = 7 macaques) (a), CD8 depletion alone (n = 7 macaques) (b) or CD8 depletion combined with N-803 (n = 7 macaques) (c). Data are mean ± s.e.m. Two-sided Wilcoxon signed-rank tests were used to compare values after the intervention to the pre-intervention baseline.

Extended Data Fig. 5 Level of virus reactivation correlated with the absence of CD8+ T cells.

a, b, Correlation between CD8+ T cell counts and viral load (SIV RNA copies per ml of plasma) on day 0, day 3, and weekly up to week 6 after interventions. a, CD8 depletion alone (n = 103 samples from 14 macaques). b, CD8 depletion combined with N-803 treatment (n = 112 samples from 14 macaques). c, d, The area under the curve (AUC) and the average pre-intervention viral load after CD8 depletion alone (c; n = 14 macaques) or CD8 depletion with N-803 treatment (d; n = 14 macaques). Correlation coefficients are calculated using the Spearman’s rank-order correlation (two-tailed, no adjustments). eg, Longitudinal viral loads (top) and CD8+ T cell counts (bottom) after N-803 treatment alone (e; n = 7 macaques), CD8 depletion alone (f; n = 14 macaques) or CD8 depletion combined with N-803 treatment (g; n = 14 macaques). Colour keys along the bottom indicate animal IDs.

Extended Data Fig. 6 HIV DNA, HIV RNA and human T cell activation levels in HIV-infected, ART-suppressed BLT humanized mice treated with N-803, CD8 depletion alone or combined CD8 depletion with N-803 .

a, b, HIV-infected, ART-suppressed BLT humanized mice were treated with N-803 (green, n = 4 BLT humanized mice), CD8 depletion alone (blue, n = 4), or treated with CD8 depletion with N-803 (red, n = 4). After 7 days, total HIV DNA (a) and cell-associated HIV RNA (b) were extracted from mononuclear cells isolated from the spleen, human-derived thymus (huThy) and lymph node (LN; HIV RNA only). cf, Percentages of HLA-DR+, CD38+, CD25+ or HLA-DR+CD38+ cells were measured in human CD4+ (c, e) or CD8+ (d, f) T cells isolated from the spleen (c, d) or human-derived thymus (e, f) of HIV-infected, ART-suppressed BLT humanized mice 7 days after treatment with N-803 (green, n = 4), CD8 depletion alone (blue, n = 4) or CD8 depletion combined with N-803 treatment (red, n = 4). Treatment groups were compared using a two-tailed Student’s t-test (a) or a Kruskal–Wallis test with a false-discovery rate correction (bf). Data are mean ± s.e.m.

Extended Data Fig. 7 Phylogenetic trees of longitudinal SGA-derived Env amino acid sequences.

Phylogenetic trees were generated for six macaques that received CD8 depletion with N-803 using Env sequences from the peak viral load (red), pre-ART (blue) and reactivation (green) time points. The Env sequence of the SIVmac239 clone used for infection is included in each tree (black). The horizontal bar below each tree indicates the genetic distance. Sequence clusters that are supported by bootstraps greater than 80% are indicated by an asterisk. Env sequences that contain a stop codon are indicated by an arrow.

Extended Data Fig. 8 Longitudinal Env amino acid divergence from the input virus and relationship with viral load in the plasma.

The number of amino acid differences between the infecting viral clone SIVmac239 and each SGA amplicon was determined using Geneious. a, Violin plots show the frequency distribution of the number of amino acid differences between sequences at each time point in each macaque. The solid line indicates the median number of amino acid differences for each individual Env sequence; the dotted lines indicate the quartiles. Peak viral load (VL) (red), pre-ART (blue) and reactivation (green) time points are shown. The animal ID and the three time points are indicated on the x axis. Statistical differences between time points for each macaque were determined by performing multiple comparisons using a Kruskal–Wallis test with Dunn’s correction. b, The average number of sequence differences for each macaque at the reactivation time point is plotted on the y axis, and the corresponding plasma viral loads are plotted on the x axis on a log10 scale. Correlation coefficients are calculated using the Spearman’s rank-order correlation (two-tailed, no adjustments).

Extended Data Fig. 9 Highlighter plots of longitudinal SGA-derived Env amino acid sequences.

Highlighter plots were generated for six representative macaques that were depleted of CD8 and treated with N-803 using Env sequences from peak VL (red box), pre-ART (blue box) and reactivation (green box) time points. The Env sequence of the SIVmac239 clone used for infection is included as the master (reference) sequence in each plot. The position of N-linked glycosylation sites on the master sequence are indicated by pink circles. Each tick represents an amino acid difference from the master sequence, as indicated in the key. Blue diamonds indicate the loss of an N-linked glycosylation site.

Extended Data Table 1 Viral loads from macaque and humanized mouse studies

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McBrien, J.B., Mavigner, M., Franchitti, L. et al. Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells. Nature 578, 154–159 (2020). https://doi.org/10.1038/s41586-020-1946-0

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