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Platelets from HIV-infected individuals on antiretroviral drug therapy with poor CD4+ T cell recovery can harbor replication-competent HIV despite viral suppression

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Science Translational Medicine  18 Mar 2020:
Vol. 12, Issue 535, eaat6263
DOI: 10.1126/scitranslmed.aat6263
  • Fig. 1 Detection of HIV in platelets from HIV-infected individuals on ART.

    (A) Number of HIV long terminal repeat (LTR) copies per million platelets in platelet-rich plasma samples from ART-treated HIV-infected individuals with viral loads either below the LOD or detectable at the date of sampling. Platelet-rich plasma from healthy donors was used as a negative control. Platelet samples in which HIV was detected are shown in orange, and samples negative for HIV are shown in gray. The number of different individuals tested (n) is shown below the graph. Data are the result of seven independent experiments. Boxplots represent medians with 25th and 75th percentiles and with minimum/maximum values indicated by whiskers (Mann-Whitney U test). (B) Top: HIV RNA+/p24-Gag+ platelets were measured by FISH–flow cytometry. Bottom left: Boxplot shows percentage of HIV RNA+/p24-Gag+ platelets from healthy donors or HIV-infected individuals on ART detected by FISH–flow cytometry and expressed as a percentage of total platelets in the platelet-rich plasma samples. Normalized percentage values are indicated in italics on the right-hand axis. The quantification threshold (orange dashed line) was established on the basis of healthy donor samples. Samples from HIV-infected individuals on ART were grouped according to the absence (gray, negative) or presence (orange, positive) of HIV in platelets (P < 0.001). The number of different individuals tested (n) is shown below the graph. Bottom right: Boxplot shows percentages converted into HIV RNA+/p24-Gag+ platelets per million platelets after data normalization and after subtracting the maximum value obtained from healthy donor control samples. The number of different individuals tested (n) is shown below the graph. Results are from seven independent experiments and are expressed as boxplots generated separately for negative and positive groups. Boxplots represent medians with 25th and 75th percentiles and with minimum/maximum values indicated by whiskers (Mann-Whitney U test). (C) Representative confocal microscopy images (top panels) show platelets from HIV-infected individuals on ART immunostained for CD41 (green) and p24-Gag (magenta). Images reconstructed in three dimensions are shown in the bottom panels. White arrowheads indicate HIV contained within platelets. Representative images are shown for platelets from 11 different HIV-infected individuals on ART. Scale bars, 1 μm. (D) Representative transmission electron micrograph of an HIV-containing platelet. A viral particle in a small vacuolar compartment is indicated by the magenta box, which is shown at a higher magnification in the image inset. The electron micrograph is representative of samples from five different HIV-infected individuals on ART with viral suppression. (E) Immunogold labeling (10 nm) of p24-Gag in cryosections was performed before electron microscopy to confirm the presence of HIV within a platelet compartment (pink arrowhead). Representative image of 10 different images obtained from two different HIV-infected individuals on ART with viral suppression. αG, α-granule; V, vacuole. Scale bars, 0.5 μm, 100 nm, or 50 nm. (F) p24-Gag immunofluorescence intensities for platelets from healthy donors or HIV-infected individuals on ART detected by flow cytometry. Results are from seven independent experiments and are expressed as boxplots generated separately for negative and positive groups. Boxplots represent median with 25th and 75th percentiles and with minimum/maximum values indicated by whiskers (Student’s t test). (G) Image shows green fluorescent protein–positive (GFP+) reporter T cells (green) after interaction with platelet-rich plasma from an HIV-infected individual on ART. Nuclei are labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue); the fluorescence signal (green) was merged with phase contrast (white arrowheads). Scale bar, 10 μm. Boxplot shows the quantification of infectious units per million platelets in platelet-rich plasma samples from HIV-infected individuals on ART who either displayed viremia below LOD or had detectable viral load at the date of sampling. Samples lacking infectious virus are in gray (negative), and those with infectious virus are in orange (positive). The number of different individuals tested (n) is shown below the graph. Results are from eight independent experiments and are expressed as boxplots generated separately for negative and positive groups. Boxplots represent medians with 25th and 75th percentiles and with minimum/maximum values indicated by whiskers (Mann-Whitney U test). ns, not significant.

  • Fig. 2 Transfer of HIV from platelets to macrophages in vitro.

    (A and B) Representative confocal microscopy images of HIV-containing platelets interacting with macrophages (MΦ) in vitro after immunostaining for CD41 (green) and p24-Gag (pink); image is merged with phase-contrast image. White arrowheads show labeled virus within platelets. Image insets show three-dimensional reconstructions or projections in xy, yz, and xz dimensions for HIV-containing platelets. Images are representative of five different individuals. Scale bars, 5 μm (main and inset figures). (C) Number of integrated copies of HIV DNA per 106 macrophages after 7 days of coculture with platelets from HIV-infected individuals on ART with viral suppression in the presence (solid orange circle) or absence (empty orange circle) of abciximab. Culture with cell-free HIV-1 was used as a positive control (magenta circle), and healthy donor platelets were used as a negative control (gray circle). (D) Infectious units produced per 106 macrophages after 7 days of coculture with platelets from HIV-infected individuals on ART with viral suppression in the presence (solid orange circle) or absence (empty orange circle) of abciximab. Culture with cell-free HIV-1 was used as a positive control (magenta circle), and healthy donor platelets were used as a negative control (gray circle). Results for (C) and (D) are presented as medians (circles) with 95% CIs (whiskers). Data represent three independent experiments with samples from five different HIV-infected individuals on ART. Mann-Whitney U test was applied for paired experimental data for platelets from HIV-infected individuals on ART, treated or not treated with abciximab in vitro.

  • Fig. 3 Platelets containing HIV correlate with low CD4+ T cell counts.

    (A) Comparison of CD4+ T cell counts at the date of sampling for HIV-infected individuals on ART whose platelets either contained (positive, orange) or did not contain (negative, gray) HIV. HIV was detected by HIV LTR qPCR, FISH flow cytometry (HIV RNA+/p24-Gag+), p24-Gag flow cytometry, and a reporter T cell assay in seven, five, seven, and eight independent experiments, respectively. HIV-infected individuals on ART with HIV-containing platelets (orange) showed lower CD4+ T cell counts (<350/μl) than did those with platelets lacking HIV (gray). Results are shown as boxplots generated separately for positive and negative groups. The Mann-Whitney U test was used to compare negative (gray) and positive (orange) groups for each HIV detection technique. (B) PCA of samples from HIV-infected individuals on ART categorized according to the presence (positive, orange) or absence (negative, gray) of HIV in platelets. These data and the clinical characteristics for each HIV-infected individual at the date of sampling are represented as variable vectors (arrows).

  • Fig. 4 Platelets containing HIV correlate with poor immunological recovery.

    (A) CD4+ T cell counts (historical nadir) for HIV-infected individuals on ART whose platelets either did (orange, positive) or did not (gray, negative) contain HIV. HIV-infected individuals on ART with HIV-containing platelets had a lower mean CD4+ T cell count nadir (<200 cells/μl) compared to the negative group (P = 0.006, Mann-Whitney U test). The number of individuals (n) is shown below the graph. (B) Mean CD4+ T cell counts for HIV-infected individuals on ART who were positive (orange line) or negative (gray line) for the presence of HIV in platelets. Data are shown for the time period of 18 months before and 18 months after the date of sampling. Analysis was first performed for a subgroup of 35 HIV-infected individuals on ART who were selected irrespective of viremia (left). This group was then further divided into 25 HIV-infected individuals on ART with controlled viremia (>1 year with viral load consistently below LOD; middle) and 10 HIV-infected individuals on ART with uncontrolled viremia (with a detectable viral load at the date of sampling or less than 12 months of continuous viral suppression; right). Red dotted line indicates a CD4+ T cell count threshold of 350 cells/μl. (C) Percentage of HIV-infected individuals on ART in immunological failure (<350 CD4+ T cells/μl after at least 1 year of follow-up including the date of sampling) grouped according to the presence (positive, orange) or absence (negative, gray) of HIV in platelets. OR, odds ratio; Fisher’s exact test.

  • Table 1 HIV-infected individuals selected for retrospective and prospective analysis of immunological status, viremia, and viral suppression.

    Patients were classified as positive or negative for the presence of HIV in platelets according to at least two different techniques. Only HIV-infected individuals for whom clinical data were available 18 months before the date of sampling were included in the analysis. The period of viral suppression in months before the date of sampling is indicated. Competent immunological status was defined as >350 CD4+ T cells/μl of blood for two or more consecutive measurements over 1 year including date of sampling for detecting HIV in platelets. Immunological failure was defined as <350 CD4+ T cells/μl blood for two or more consecutive measurements over 1 year including date of sampling for detecting HIV in platelets. Controlled viremia was defined as <1.60 HIV RNA log10 copies/ml of plasma for at least 1 year before sampling. Uncontrolled viremia was defined as >1.60 HIV RNA log10 copies/ml of plasma at the date of sampling or less than 12 months of continuous viral suppression.

    Individual numberImmunological statusViremiaMonths suppressed before
    sampling
    Individuals negative for HIV
    in platelets
    39CompetentControlled17
    32CompetentControlled18
    75CompetentControlled31
    54CompetentControlled18
    52CompetentControlled12
    11CompetentControlled18
    51CompetentControlled18
    16CompetentControlled12
    15CompetentControlled13
    61CompetentControlled13
    66CompetentControlled47
    67CompetentControlled31
    4CompetentControlled78
    56CompetentControlled62
    53In failureControlled22
    59In failureControlled18
    Median months under controlled viremia (CI)18 (15–31)
    7In failureUncontrolled<12
    60CompetentUncontrolled<12
    24CompetentUncontrolled<12
    46CompetentUncontrolled<12
    Individuals positive for HIV
    in platelets
    9In failureControlled24
    27In failureControlled38
    48In failureControlled19
    6In failureControlled20
    49In failureControlled50
    64In failureControlled52
    79In failureControlled14
    80In failureControlled62
    37CompetentControlled13
    Median months under controlled viremia (CI)24 (14–52)
    74CompetentUncontrolled<12
    25CompetentUncontrolled<12
    18In failureUncontrolled<12
    78In failureUncontrolled<12
    40In failureUncontrolled<12
    41In failureUncontrolled<12

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/535/eaat6263/DC1

    Material and Methods

    Fig. S1. Platelet-rich plasma preparations from HIV-infected individuals were not contaminated by HIV-infected CD4+ T cells.

    Fig. S2. Flow cytometry gating strategy for the detection of HIV in platelets from HIV-infected individuals on ART.

    Fig. S3. HIV-containing platelets detected by electron and confocal microscopy.

    Fig. S4. Infection of macrophages by HIV in platelets is due to direct interactions and not residual cell-free viruses present in platelet-rich plasma.

    Fig. S5. HIV-1 integrated proviral DNA in megakaryocytes from HIV-infected individuals on ART with viral suppression.

    Fig. S6. Deep sequencing and phylogenetic analysis of HIV env V3 segments before and after viral suppression in the same HIV-infected individuals on ART.

    Fig. S7. Interaction between platelets and macrophages is blocked by the anti–integrin αII/β3 Fab abciximab.

    Fig. S8. Detection of HIV-containing platelets does not correlate with other clinical characteristics.

    Table S1. Clinical information on HIV-infected individuals on ART analyzed for HIV in platelets.

    Table S2. Clinical information on each HIV-infected individual on ART analyzed for HIV in platelets.

    Table S3. Available viral load measurements for HIV-infected individuals on ART positive for HIV in platelets and with a viral load below the LOD at the date of sampling.

    Table S4. Clinical information for each HIV-infected individual on ART analyzed for HIV in megakaryocytes.

    Table S5. ART regimens for HIV-infected individuals classified as positive or negative for the presence of HIV in platelets.

    Table S6. CD4+ T cell counts and viral load measurements for HIV-infected individuals on ART with controlled viremia and positive for HIV in platelets.

    Table S7. List of Gag mRNA probe sequences used for FISH–flow cytometry.

    Data file S1. Individual-level data for Figs. 1, 2, and 4 and figs. S1, S4, and S7.

    Movie S1. Laser scanning confocal microscopy image of an HIV-containing platelet being engulfed by a macrophage.

    References (4968)

  • The PDF file includes:

    • Material and Methods
    • Fig. S1. Platelet-rich plasma preparations from HIV-infected individuals were not contaminated by HIV-infected CD4+ T cells.
    • Fig. S2. Flow cytometry gating strategy for the detection of HIV in platelets from HIV-infected individuals on ART.
    • Fig. S3. HIV-containing platelets detected by electron and confocal microscopy.
    • Fig. S4. Infection of macrophages by HIV in platelets is due to direct interactions and not residual cell-free viruses present in platelet-rich plasma.
    • Fig. S5. HIV-1 integrated proviral DNA in megakaryocytes from HIV-infected individuals on ART with viral suppression.
    • Fig. S6. Deep sequencing and phylogenetic analysis of HIV env V3 segments before and after viral suppression in the same HIV-infected individuals on ART.
    • Fig. S7. Interaction between platelets and macrophages is blocked by the anti–integrin αII/β3 Fab abciximab.
    • Fig. S8. Detection of HIV-containing platelets does not correlate with other clinical characteristics.
    • Table S1. Clinical information on HIV-infected individuals on ART analyzed for HIV in platelets.
    • Table S2. Clinical information on each HIV-infected individual on ART analyzed for HIV in platelets.
    • Table S3. Available viral load measurements for HIV-infected individuals on ART positive for HIV in platelets and with a viral load below the LOD at the date of sampling.
    • Table S4. Clinical information for each HIV-infected individual on ART analyzed for HIV in megakaryocytes.
    • Table S5. ART regimens for HIV-infected individuals classified as positive or negative for the presence of HIV in platelets.
    • Table S6. CD4+ T cell counts and viral load measurements for HIV-infected individuals on ART with controlled viremia and positive for HIV in platelets.
    • Table S7. List of Gag mRNA probe sequences used for FISH flow cytometry.
    • Legend for movie S1
    • References (4968)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Laser scanning confocal microscopy image of an HIV-containing platelet being engulfed by a macrophage.
    • Data file S1 (Microsoft Excel format). Individual-level data for Figs. 1, 2, and 4 and figs. S1, S4, and S7.

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