Persistence of hepatitis C virus in peripheral blood mononuclear cells of patients who achieved sustained virological response following treatment with direct-acting antivirals is associated with a distinct pre-existing immune exhaustion status

Abstract

Hepatitis C virus (HCV) is a primary hepatotropic pathogen responsible for acute and chronic hepatitis C, however, it can also cause "occult" infection (OCI), defined as the presence of the virus' genetic material in hepatocytes and/or peripheral blood cells, but not in plasma/serum. Assessment of the sustained virologic response (SVR) after treatment with direct-acting antivirals (DAA) is based exclusively on HCV-RNA testing in plasma/serum, which may preclude the diagnosis of post-treatment OCI. Possible clinical consequences of OCI were described previously, but its occurrence after DAA-based antiviral treatment programs and determinants of the virus persistence are not fully elucidated. The aim of this study was to assess the incidence of post-treatment OCI after successful DAA-based treatment and to identify clinical and immunological factors associated with this phenomenon. In 97 patients treated with DAA, HCV-RNA was tested by RT-PCR in peripheral blood mononuclear cells (PBMC) at baseline (i.e., before the onset of treatment) and at the time of SVR assessment. Before treatment, HCV-RNA was detectable in all patients' PBMC. All subjects responded to therapy according to the clinical criteria, but 9 (9.3%) patients revealed the HCV-RNA in PBMC at SVR. In most of these cases, post-DAA OCI was related to switch of the dominant infecting genotype. Post-treatment OCI was characterized by significantly lower pre-treatment HCV viral load and lower expression of Tim-3 (T-cell immunoglobulin and mucin domain-containing protein 3) on CD8⁺ T-cells. Our results imply that post-treatment OCI may be related to lower pretreatment viral load as well as distinct pre-existing immune exhaustion status.

Keywords: Direct-acting antivirals (DAA); Immune exhaustion; Occult HCV infection; Sustained virologic response (SVR).

Fig. 1

Baseline age (A), BMI (B), initial bilirubin levels (C), ALT activity (D), viral load (E), percentage of CD4⁺ (F) and CD8⁺ (G) T-cells in patients with undetectable (NO OCI) and detectable (OCI) HCV-RNA in PBMC after DAA treatment. Each point represents a single result, whiskers represent range, horizontal lines represent the median. P-value representing statistically significant difference in pairwise comparisons is indicated above the line.

Fig. 2

Flow cytometry analysis of percentages of CD4⁺ and CD8⁺ T-cells per total T-cells (i.e., CD3⁺) with membrane expression of PD-1 (A), Tim-3 (B) and PD-1 + Tim-3 (C) in patients with undetectable (NO OCI) and detectable (OCI) HCV-RNA in PBMC after DAA treatment. Each point represents a single result, whiskers represent range, horizontal lines represent the median. P-value representing statistically significant difference in pairwise comparisons is indicated above the line.

Fig. 3

Baseline and post-treatment levels of IL-10 (A) sPD-1 (B), sTim-3 (C) and sLAG-3 (D) in plasma in patients with undetectable (NO OCI) and detectable (OCI) HCV-RNA in PBMC after DAA treatment. Each point represents a single result, whiskers represent range, horizontal lines represent the median. P-value representing statistically significant difference in pairwise comparisons is indicated above the line. Pre-TX, before treatment, Post-TX, post-treatment.