Immune phenotype and function of natural killer and T cells in chronic hepatitis C patients who received a single dose of anti-MicroRNA-122, RG-101

Abstract

MicroRNA-122 is an important host factor for the hepatitis C virus (HCV). Treatment with RG-101, an N-acetylgalactosamine-conjugated anti-microRNA-122 oligonucleotide, resulted in a significant viral load reduction in patients with chronic HCV infection. Here, we analyzed the effects of RG-101 therapy on antiviral immunity. Thirty-two chronic HCV patients infected with HCV genotypes 1, 3, and 4 received a single subcutaneous administration of RG-101 at 2 mg/kg (n = 14) or 4 mg/kg (n = 14) or received a placebo (n = 2/dosing group). Plasma and peripheral blood mononuclear cells were collected at multiple time points, and comprehensive immunological analyses were performed. Following RG-101 administration, HCV RNA declined in all patients (mean decline at week 2, 3.27 log10 IU/mL). At week 8 HCV RNA was undetectable in 15/28 patients. Plasma interferon-γ-induced protein 10 (IP-10) levels declined significantly upon dosing with RG-101. Furthermore, the frequency of natural killer (NK) cells increased, the proportion of NK cells expressing activating receptors normalized, and NK cell interferon-γ production decreased after RG-101 dosing. Functional HCV-specific interferon-γ T-cell responses did not significantly change in patients who had undetectable HCV RNA levels by week 8 post-RG-101 injection. No increase in the magnitude of HCV-specific T-cell responses was observed at later time points, including 3 patients who were HCV RNA-negative 76 weeks postdosing.

Conclusion: Dosing with RG-101 is associated with a restoration of NK-cell proportions and a decrease of NK cells expressing activation receptors; however, the magnitude and functionality of ex vivo HCV-specific T-cell responses did not increase following RG-101 injection, suggesting that NK cells, but not HCV adaptive immunity, may contribute to HCV viral control following RG-101 therapy. (Hepatology 2017;66:57-68).

Figure 1

(A) Mean change in HCV RNA levels between baseline and week 4 in patients dosed with placebo (black dots), 2 mg/kg (red dots) and 4 mg/kg (blue dots) RG-101. (B) Plasma IP-10 levels in healthy controls (HC) and CHC patients (HCV) at baseline, bars indicate median. (C) Change in plasma IP-10 levels (median) in patients dosed with placebo (black dots), 2 mg/kg (red dots) and 4 mg/kg (blue dots) RG-101, level of significant difference indicated between RG-101 dosed (2 mg/kg and 4 mg/kg) and placebo dosed patients. (D) Change in plasma IP-10 levels between baseline and week 1 in patients with HCV RNA levels < LLOQ and > LLOQ at week 8, bars indicate median. Statistical testing; T-test (A) and Mann–Whitney test (B-D).

Figure 2

(A) Proportion of NK cells as a percentage of total lymphocytes in healthy controls (HC) and CHC patients dosed with RG-101 at baseline (bl), week (wk) 2, 8 and 12-28 (including patients with viral rebound between week 12 and 28 and patients with HCV RNA < LLOQ at week 28). Data is shown grouped in patients who are HCV RNA positive or have HCV RNA levels < LLOQ, as well as in placebo dosed patients, bars indicate median. (B) Proportion of CD56^bright and CD56^dim cells as a percentage of total NK cells in RG-101 dosed patients at baseline(bl), week (wk) 2, 8, and 12-28, bars indicate median. Representative dot plot showing NK cell gating in total lymphocyte population. CD56^bright and CD56^dim NK cell gates are shown (C) Percentage of TRAIL+ cells within CD56^bright NK cells in healthy controls and patients dosed with RG-101, bars indicate median. Patients who had HCV RNA levels < LLOQ are depicted in green dots, patients who had HCV RNA levels > LLOQ in red. (D) Representative dot plot showing TRAIL gating, percentages of TRAIL positive cells within the CD56^bright NK cell population (black) are shown. CD56^dim NK cells are shown in grey for comparison. Statistical testing; Mann–Whitney test (A,C) and Wilcoxon matched pairs test (B,C).

Figure 3

Proportion of (A) CD16, (B) NKp30, (C) NKp46, (D) T-bet, (E) Eomes and (F) Ki67 in healthy controls (HC) as well as in CHC patients dosed with RG-101 (n=28) at baseline (bl), week (wk) 2 and 8, bars indicate median. Statistical testing; Mann–Whitney test and Wilcoxon matched pairs test (A-F). Patients who have HCV RNA levels < LLOQ are shown in green dots, patients with HCV RNA levels > LLOQ are in red dots.

Figure 4

(A) Ex vivo multimer positive cells as a proportion of total CD8+ T cells in RG-101 dosed patients at baseline (bl), week (wk) 2, wk 8 (including n=2 HCV RNA <LLOQ, excluding n=2 due to missed visit). Each dot represents one epitope, a positive response was defined as > 5 events with a lower threshold of 10,000 measured CD8+ T cells, bars indicate median. (B) ELISpot responses at baseline and week 8 in patients dosed with RG-101 (left) and placebo (right). Patients’ PBMCs were stimulated ex vivo with genotype specific HCV-peptide pools. (C) ELISpot responses at baseline and week 8 (and week 76) in patients dosed with RG-101 with HCV RNA levels > LLOQ (left) and < LLOQ (right) at week 8. (D) Change between baseline and week 8 ELISpot responses in patients dosed with RG-101 with HCV RNA levels > LLOQ (green dots) and < LLOQ (blue dots) at week 8 as well as placebo dosed patients, bars indicate median. Statistical testing; Mann–Whitney test (D) and Wilcoxon matched pairs test (A-C).

Figure 5

ELISpot responses (left y-axis) and viral load (right y-axis) in patients who were HCV RNA negative for 76 weeks (upper graphs, patients #1-3) as well as in patients who had HVC RNA levels < LLOQ for 20 or more weeks (lower graphs, patients #4-8). (I): patient #4 had < 1 log10 increase in HCV RNA at week 20 (PBMC missing), and was negative again at week 24. Rebound was at week 28. (II): patient #5 had > 1 log10 increase in HCV RNA at week 24 and < 1 log10 increase in HCV RNA at retest (week 26, last available PBMCs), rebound was at week 36.