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Research Article

Brain Macrophages in Simian Immunodeficiency Virus-Infected, Antiretroviral-Suppressed Macaques: a Functional Latent Reservoir

Claudia R. Avalos, Celina M. Abreu, Suzanne E. Queen, Ming Li, Sarah Price, Erin N. Shirk, Elizabeth L. Engle, Ellen Forsyth, Brandon T. Bullock, Feilim Mac Gabhann, Stephen W. Wietgrefe, Ashley T. Haase, M. Christine Zink, Joseph L. Mankowski, Janice E. Clements, Lucio Gama
Arturo Casadevall, Editor
Claudia R. Avalos
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Celina M. Abreu
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Suzanne E. Queen
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Ming Li
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Sarah Price
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Erin N. Shirk
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Elizabeth L. Engle
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Ellen Forsyth
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Brandon T. Bullock
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Feilim Mac Gabhann
e Department of Biomedical Engineering and Institute for Computational Medicine, Johns Hopkins University, Baltimore, Maryland, USA
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Stephen W. Wietgrefe
d Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA
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Ashley T. Haase
d Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, Minnesota, USA
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M. Christine Zink
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
b Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Joseph L. Mankowski
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
b Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
c Department of Neurology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Janice E. Clements
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
b Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
c Department of Neurology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Lucio Gama
a Department of Molecular and Comparative Pathobiology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA
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Arturo Casadevall
Johns Hopkins Bloomberg School of Public Health
Roles: Editor
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DOI: 10.1128/mBio.01186-17
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  • FIG 1 
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    FIG 1 

    Viral load in plasma and CSF of two cohorts of SIV-infected ART-treated macaques. Two cohorts of SIV-infected pig-tailed macaques were treated with similar ART regimens (listed above the horizontal green line). (A and B) Macaques in cohort A1 were suppressed for more than 500 days and were treated with LRAs (ingenol-B, orange; ingenol-B plus suberoylanilide hydroxamic acid [SAHA], purple). (C and D) Macaques in cohort A2 followed similar protocol as those in cohort A1 but were suppressed for 100 to 400 days and treated with LRAs (ingenol plus SAHA in purple) in the last 60 days before euthanasia (Table 1). Median values for each group of animals are depicted in red for plasma (A and C) and dark blue for CSF (B and D). Analyses of samples with values below the limit of detection for the SIV qPCR assay (100 SIV RNA copies/ml) were repeated using ddPCR.

  • FIG 2 
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    FIG 2 

    Analyses of viral decay in plasma and CSF. The line for each cohort represents the curve best fitting the geometric means of longitudinal viral loads (symbols) for that cohort in plasma (A) and CSF (B). Values in the insets show half-lives (t1/2) and R2 values for phase (ph) 1 and phase 2 in the curves. Cohort A0 represents data previously published (34).

  • FIG 3 
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    FIG 3 

    Quantitation of latently infected BrMΦ in ART-treated macaques by MΦ-QVOA. (A and B) Quantitation of infected BrMΦ from two groups of ART-treated macaques. In panel B, wells containing <50 SIV RNA copies/ml were also considered positive, and IUPM (infectious units per million cells) values were calculated accordingly. Boxes in panels A and B indicate values under the limit of detection, i.e., BrMΦ samples that did not present positive well results by qPCR. (C) Comparison between the numbers of SIV-infected BrMΦ isolated from animals that were not given ART (−ART) and the numbers isolated from animals that were treated with ART and showed full suppression (+ART; PmA13 not included). The horizontal black line represents the median IUPM values. The MΦ-QVOA results from SIV-infected animals without ART were previously reported (35). Significance was determined by Mann-Whitney nonparametric t test; a P of <0.05 was considered significant. (D) Numbers of SIV RNA copies in the supernatants of QVOA-positive wells separated according to the pattern of replication (<50 copies/ml, >50 copies/ml without spread, or replicative).

  • FIG 4 
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    FIG 4 

    Virus from BrMΦ QVOA is replication competent in PBMCs. The graph depicts the increased levels of SIV RNA in supernatants of PBMC cultures that were subjected to spinoculation with 100 µl of supernatants collected from BrMΦ QVOA wells. Different wells from each of the five analyzed macaques are depicted in the same color, and each line represents the result of one supernatant transfer. The first time point depicts the number of SIV RNA copies contained in the 100-µl volume of the BrMΦ QVOA well that was used for spinoculation. The other time points show the numbers of total supernatant copies measured at 5 to 7 days and 10 to 14 days postspinoculation.

  • FIG 5 
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    FIG 5 

    In situ hybridization (ISH) for SIV RNA in brain sections. Data are representative of ISH results of analysis of SIV RNA in brain (occipital cortex) of macaque PmA13 (top row) and Mn3, which represents a previously published SIV-infected macaque that discontinued ART and had SIV rebound (lower row) (36); magnification, ×40.

Tables

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  • TABLE 1 

    Description of macaque cohorts, the antiretroviral therapy regimen, and levels of CD4+ T cells, monocytes, plasma viral load, and CSF viral load at the terminal time pointa

    Cohort and animal identifierART (no. of days)Cell counts (no. of cells/µl blood)Viral load (no. of SIV RNA copies/ml)Comment
    CD4+ T cellsMonocytesPlasmaCSF
    A1
        PmA116154741,092<10<10NA
        PmA126161,074485<10<10Treated with LRAs after 550 dpi
        PmA136053201,3461,80018,000Treated with LRAs after 550 dpi
    A2
        PmA21394280340<10<10NA
        PmA223913242,310<10<10Treated with LRAs after 242 dpi
        PmA23345331410<10<10Treated with LRAs after 249 dpi
        PmA24182291350<10<10NA
        PmA25182790310<10<10NA
    • ↵ a The antiretroviral therapy (ART) regiment for both cohort A1 and cohort A2 consisted of tenofovir, darunavir, integrase inhibitor L000870812, and ritonavir. LRA, latency reverse agents; dpi, days postinfection; NA, not applicable.

  • TABLE 2 

    SIV DNA and RNA levels in two brain regions (basal ganglia and parietal cortex) at terminal point and number of latently infected BrMΦ for each macaquea

    Cohort and animal identifierNo. of SIV DNA copies per 106 cellsNo. of SIV RNA copies per µg RNABrain MΦ-QVOA IUPM
    Basal gangliaParietal cortexBasal gangliaParietal cortexWithout wells (<50 copies/ml)With wells (<50 copies/ml)
    A1
        PmA11<10<10<10<100.1470.917
        PmA12<10<10<10<100.0360.230
        PmA13<1064<10<100.0360.110
    A2
        PmA21<10<10<10<101.4451.445
        PmA22<10<10<10<100.1771.309
        PmA23<10<10<10<107.3627.362
        PmA24<10<10<10<10<0.021<0.021
        PmA25<10<10<10<100.6130.613
    • ↵ a QVOA values are presented and represent the presence or absence of positive wells with fewer than 50 SIV RNA copies/ml of supernatant. Values reflect maximum likelihood estimates of infection frequency in IUPM (infectious units per million cells). Given the resolution of the assay, the 95% confidence interval is typically 0.2 to 4 times the reported value.

  • TABLE 3 

    Quantitation of infected CD4+ T cells in the MΦ-QVOA using detection of TCRβ RNA, as described by Avalos et al. (35)

    Cohort and animal identifierTCRβ in MΦ-QVOA (no. of RNA copies/105 cells)% CD3+ T cells in MΦ-QVOA by TCRβ% CD4+ T cells in blood CD3+ T cells% CD4+ T cells in MΦ-QVOACD4+ T cell IUPM by PBMC QVOANo. of infected CD4+ T cells per 106 QVOA cells
    A1
        PmA113,8710.9750.90.501.30.0065
        PmA120060.200.300
        PmA1320,4285.1135.11.800.400.0072
    A2
        PmA210055.200.050
        PmA220056.800.070
        PmA230056.400.050
        PmA240045.800.010
        PmA250048.900.060
  • TABLE 4 

    SIV DNA and RNA levels in BrMΦ isolated from basal ganglia and parietal cortex of SIV-infected ART-treated macaquesa

    Cohort and animal identifierSIV DNA (no. of copies per 106 BrMΦ)SIV RNA (no. of copies per 106 BrMΦ)CSF (no. of SIV RNA copies/ml)Basal ganglia (no. of SIV RNA copies/µg total RNA)MΦ-QVOA (no. of infected BrMΦ per million cells)
    A1
        PmA1120<5<10<100.92
        PmA129<5<10<100.23
        PmA1311933,98418,000<100.11
    A2
        PmA2140<5<10<101.45
        PmA2260<5<10<101.31
        PmA231,606230<10<107.36
        PmA24574,652<10<10<0.02
        PmA25671,263<10<100.61
    • ↵ a SIV RNA levels in CSF and basal ganglia and no. of infected BrMΦ evaluated at necropsy are also presented for comparison.

  • TABLE 5 

    Number of SIV RNA-positive foci per gram of brain tissue estimated by in situ hybridization

    Cohort and animal identifierArea analysed (μm2)No. of positive foci per analysed fieldDensity of SIV+ cells per gram of tissue
    A1
        PmA111.41 × 1080, 1, 0, 0, 0, 0, 0202
        PmA122.54 × 1080, 0, 0, 0, 0, 0, 0<102
        PmA131.16 × 1081, 1, 1, 1, 1, 3, 12,220
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Brain Macrophages in Simian Immunodeficiency Virus-Infected, Antiretroviral-Suppressed Macaques: a Functional Latent Reservoir
Claudia R. Avalos, Celina M. Abreu, Suzanne E. Queen, Ming Li, Sarah Price, Erin N. Shirk, Elizabeth L. Engle, Ellen Forsyth, Brandon T. Bullock, Feilim Mac Gabhann, Stephen W. Wietgrefe, Ashley T. Haase, M. Christine Zink, Joseph L. Mankowski, Janice E. Clements, Lucio Gama
mBio Aug 2017, 8 (4) e01186-17; DOI: 10.1128/mBio.01186-17

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Brain Macrophages in Simian Immunodeficiency Virus-Infected, Antiretroviral-Suppressed Macaques: a Functional Latent Reservoir
Claudia R. Avalos, Celina M. Abreu, Suzanne E. Queen, Ming Li, Sarah Price, Erin N. Shirk, Elizabeth L. Engle, Ellen Forsyth, Brandon T. Bullock, Feilim Mac Gabhann, Stephen W. Wietgrefe, Ashley T. Haase, M. Christine Zink, Joseph L. Mankowski, Janice E. Clements, Lucio Gama
mBio Aug 2017, 8 (4) e01186-17; DOI: 10.1128/mBio.01186-17
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KEYWORDS

brain
macrophages
Simian Acquired Immunodeficiency Syndrome
simian immunodeficiency virus
Virus Latency
brain
human immunodeficiency virus
latency
macrophages
simian immunodeficiency virus

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