IFNα and its signature are increased in plasma of HIV-1-infected subjects not receiving ART.

<p>Plasma IFN-I bioactivity measured by the iLite™ bioassay was increased in plasma of HIV-1-infected subjects in comparison to uninfected subjects (median 1.04 IFNα2 equivalent units for HIV-1-infected subjects, median 0.89 IFNα2 equivalent units for uninfected subjects, p = 0.012) (A). IFNα was increased in plasma of HIV-1-infected subjects in comparison to uninfected subjects (median 4.27 pg/ml for HIV-1-infected subjects, median 3.13 pg/ml for uninfected subjects, p<0.001) (B). IFNβ was not increased in plasma of HIV-1-infected subjects compared to uninfected subjects (median 2.34 pg/ml for HIV-1-infected subjects, median 2.34 pg/ml for uninfected subjects, p = 0.560) (B). IFNω was not increased in plasma of HIV-1-infected subjects compared to uninfected subjects (median 4.69 pg/ml for HIV-1-infected subjects, median 4.69 pg/ml for uninfected subjects, p = 0.837) (B). Plasma IFNα levels were strongly associated with plasma IFN-I bioactivity in HIV-1-infected subjects (r = 0.711, p<0.001) (C). Plasma IP-10 was increased in plasma of HIV-1-infected subjects in comparison to uninfected subjects (median 538.2 pg/ml for HIV-1-infected subjects, median 132.6 pg/ml for uninfected subjects, p = 0.002) (D). Slight variations in sample sizes for different assays occur as results for some subjects were not available.</p

Therapeutic administration of IFNα induces increased expression of the activation marker CD38 on memory CD8 T cells in HCV-infected subjects.

<p>A longitudinal study was conducted to assess CD38 expression on memory (CD45RO+) CD8 T cells. Seven HCV-infected (HIV-1-uninfected) subjects were studied immediately prior to initiation of therapy with s.c. pegylated IFNα and oral ribavirin, and after 4 and 12 weeks of therapy. CD38-specific MFI (MFI for CD38 minus MFI for IgG1 isotype control) increased on memory CD8 T cells in all seven treated subjects at 4 weeks of therapy (p = 0.018). A further increase in CD38 was observed in five of seven subjects by week 12 of therapy (p = 0.018). The difference in CD38 expression between week 0 (prior to therapy) and week 12 was highly significant (p = 0.018).</p

Plasma IFNα and IFN-I bioactivity are associated with plasma HIV-1 RNA levels, CD4 T cell count and immune activation in untreated subjects with chronic HIV-1-infection.

<p>Plasma HIV-1 RNA levels in HIV-1-infected subjects without ART correlated positively with plasma IFN-I bioactivity (r = 0.329, p = 0.017) (A), as well as with plasma IFNα (r = 0.356, p = 0.008) (B). Absolute CD4 T cell count in these subjects correlated inversely with plasma IFN-I bioactivity (r = -0.426, p = 0.002) (C) and plasma IFNα (r = -0.407, p = 0.002) (D). Expression of CD38 by memory (CD45RO+) CD8 T cells in these subjects correlated positively with plasma IFN-I bioactivity (r = 0.374, p = 0.021) (E) and with plasma IFNα (r = 0.387, p = 0.01) (F).</p

Plasma IFNα and IP-10 levels are comparable between HIV-1 infected subjects on ART and uninfected subjects.

<p>IFNα and IP-10 levels were determined in the plasma of 25 individuals with chronic HIV-1 infection who received ART and responded with HIV-1 RNA below assay detection limits (<50 copies/ml) for two years or more. IFNα was detected in plasma from only one such donor, and then at trace levels (A). The difference in plasma IFNα level between HIV-1 infected subjects without ART and HIV-1-infected subjects with ART was highly significant (p<0.001). Plasma IP-10 levels in ART-treated HIV-1-infected donors were comparable to those in uninfected donors (p = 0.460) and were significantly lower in comparison to untreated HIV-1-infected donors (p<0.001) (B).</p

IFNα mRNA expression is elevated in lymph nodes, but not peripheral blood leukocytes, of HIV-1-infected subjects.

<p>There was no significant difference in expression of IFNα mRNA in whole blood leukocytes of HIV-1-infected subjects without ART (median relative expression of 0.10) and uninfected subjects (median relative expression of 0.88) (p = 0.981) (A). Similarly, there was no significant difference in IFNβ mRNA expression in whole blood leukocytes (median relative expression of 0.003 for HIV-1-infected subjects, median relative expression of 0.001 for uninfected subjects; p = 0.298) (A). An IFN-I signature was evident in peripheral blood leukocytes, as expression of the ISG MxA was significantly increased in HIV-1-infected subjects without ART compared to uninfected subjects (median relative expression of 0.85 for HIV-1-infected subjects, median relative expression of 0.26 for uninfected subjects; p = 0.016) (A). In contrast, expression of IFNα mRNA in lymph node tissue was significantly elevated in HIV-1-infected subjects without ART (median relative expression of 0.93) relative to uninfected subjects (median relative expression of 0.12) (p = 0.037) (B). There was no statistically significant difference in IFNβ mRNA expression between the two donor groups (median relative expression of 0.20 for HIV-1-infected subjects, median relative expression of 0.06 for uninfected subjects; p = 0.728) (B). Expression of the ISG MxA was significantly increased in lymph node tissue from HIV-1-infected subjects compared to uninfected subjects (median relative expression of 1.05 for HIV-1-infected subjects vs. 0.45 for uninfected subjects; p = 0.037) (B).</p

Fluorescence-activated cell sorting (FACS) strategies for PBMC samples (A-G) and GI tract samples (H-M; ileum shown as example).

<p><b> </b>For PBMC, all cells were included (panel A), doublets excluded (panel B), and residual non-viable cells excluded by LIVE/DEAD violet cell staining ("Viab dump," panel C). Low-frequency CCR5+ events were then collected in one sorting tube (inside box gate, panel D). Of the remaining, CCR5- events (outside box gate, panel D), CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel E), with remaining events collected in a second sorting tube (outside polygon gate, panel E). CCR5-CD3+ events negative for CD14 and CD11c that were also CD8- (panel F) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel G) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. For ileum and rectum, all cells were included (panel H) and then doublets excluded (panel I). Viable CD45+ events were included for further gating (inside polygon gate, panel J), with all events outside this gate collected in one sorting tube as non-hematopoietic cells. CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel K), with remaining events collected in a second sorting tube (outside polygon gate, panel K). CD3+ events negative for CD14 and CD11c that were also CD8- (panel L) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel M) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. Numbers in upper-right corners of flow plots indicate the percentages of events on plots falling inside gates shown. </p

Timeline for clinical treatments and study samples.

<p>Timeline for clinical treatments and study samples.</p

HIV-specific antibodies.

<p>Blood from four time points was tested for HIV specific antibody levels using the HIV-1/2 VITROS assay (3A), a detuned version of the HIV-1 VITROS assay (3B), and the Limiting Antigen avidity assay (3C). The y-axis shows the relative level of total HIV-specific antibody, as expressed as the signal to cutoff ratio (3A–B) or normalized optical density, ODn (3C). The x axis represents months since transplant. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003347#ppat-1003347-g003" target="_blank">Figure 3A</a>, the dotted line represents the diagnostic HIV antibody assay cut-off level used to classify individuals as HIV-positive or HIV-negative. For purposes of comparison, HIV antibody responses were also measured in HIV-uninfected adults, untreated HIV-infected adults, and ART-treated chronically-infected adults using the detuned HIV-1 VITROS assay (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003347#ppat-1003347-g003" target="_blank">Figure 3D</a>). The bars in the scatterplots represent the median and interquartile ranges of distributions of seroreactivity for each group. Finally, samples from the Berlin Patient were tested for antibodies to other infectious diseases (3E). Tests included antibodies to CMV (strong positive, above the limit of detection), EBV, measles, and hepatitis B (all within the range of detection) as well as VZV, mumps, rubella, and toxoplasmosis (all negative, below the limit of detection). Only the results within detectable range of the assay are shown. S/CO = signal/cutoff ratio; ODn = normalized optical density; AI = antibody index.</p

HIV Gag-specific cell mediated immune responses.

<p>PBMC were obtained from the Berlin Patient (solid red circles), HIV-uninfected adults (open black circles in 4A–B), chronically HIV-infected adults on long term ART with undetectable plasma viral loads (open black circles, 4C–D), and elite controllers (open black circles, 4E–F). PBMC were stimulated with CMV pp65 or HIV Gag peptide pools, and flow cytometry was used to measure the percentage of CD4+T cells (4A, 4C, 4E) or CD8+T cells (4B, 4D, 4F) with intracellular staining for interferon-γ, tumor necrosis factor-α, IL-2, or CD107. The y axis shows the percent of T cells that express each cytokine in response to HIV Gag. The solid red circle indicates the Berlin Patient, while open black circles indicate individuals from comparator groups.</p

HIV RNA in plasma.

1<p>Historical, as determined from the number of positive wells out of all wells containing samples from HIV- subjects/donors that had been processed and run along with positive samples.</p>2<p>ND = not detected.</p