Sporadic reappearance of minute amounts of hepatitis C virus RNA after successful therapy stimulates cellular immune responses

Abstract

Background & aims: Several studies have reported hepatitis C virus (HCV) RNA sequences in the circulation after treatment-induced or spontaneous recovery. We investigated whether the HCV RNA represents persistence of HCV infection or reinfection.

Methods: We studied 117 patients who recovered from HCV infection (98 following therapy and 19 spontaneously). A reverse-transcription polymerase chain reaction assay was used to detect the 5'-untranslated region of HCV. T-cell responses were studied by enzyme-linked immunospot for interferon-γ.

Results: Plasma samples from 15% of treatment-recovered patients and no spontaneously recovered patient tested positive for HCV RNA. Lymphocytes from 3 patients who responded to therapy and 1 who recovered spontaneously tested positive. The frequency of HCV RNA detection in plasma correlated inversely with the time after the end of treatment. Post-treatment HCV 5'-untranslated region sequences matched pretreatment sequences in 85% of cases. T-cell responses were significantly greater at time points with detectable trace amounts of HCV RNA than at time points without detectable HCV RNA (P = .035) and were primarily against nonstructural HCV antigens. The immune hierarchy was preserved over 5 years in patients whose post-treatment HCV RNA sequences matched pretreatment sequences, indicating HCV RNA persistence. An altered immune hierarchy with dominant immune responses, shifting from nonstructural to structural antigens, was observed in a single patient whose post-treatment HCV genotype differed from that of the pretreatment genotype, indicating HCV reinfection.

Conclusions: Trace amounts of HCV RNA of pretreatment sequence persisted and reappeared sporadically in the circulation within 8 years after recovery from hepatitis C but not thereafter, indicating that patients are cured of HCV infection. Reappearance of HCV RNA induced HCV-specific T-cell responses.

Figure 1

HCV persists for a limited time after spontaneous recovery from hepatitis C. (A) Gel electrophoresis of nested HCV 5′UTR PCR products from PBMC of a spontaneously recovered patient. Weeks represent time after initial clinical presentation. Serum samples tested negative for HCV RNA at all time points. Each PBMC sample was followed by PBMC from a healthy, HCV-uninfected blood donor during the RNA extraction, RT, and PCR steps. (B) The frequency of HCV-specific, IFN-γ-producing T cells as determined in ELISpot assays with pools of overlapping HCV peptides decreases with clearance of HCV RNA.

Figure 2

The prevalence of detectable HCV RNA decreases with time after cessation of therapy. Cross-sectional analysis of plasma samples from 95 patients by nested RT-PCR with primers specific for the HCV 5′ UTR (sensitivity, < 40 copies/mL). (A) HCV RNA is detected solely in the first 8 years after cessation of therapy. Mean and standard error of mean are indicated. (B) Step-wise decrease in the prevalence of HCV RNA positive results within 2-year periods after the end of treatment.

Figure 3

HCV RNA positive time points (solid circles) are interspersed with HCV RNA negative time points (open circles). Longitudinal analysis of prospectively collected plasma samples after cessation of treatment by nested RT-PCR (assay as in Figure 2). Patients 5, 10, 11, 19, 20, 25, 32, 34, 35, 36, 46, 84, and 97 were studied at 1–3 time points more than 10.5 years after recovery (not shown). Patient 75 experienced high-titer HCV recurrence (see legend to Table 3 for details). For patient 75, the depicted results less than 1.1 years after end of treatment are derived from serum samples tested by COBAS Amplicor HCV Test 2.0.

Figure 4

HCV-specific T-cell responses are stronger at time points with detectable HCV RNA than at those without. Prospective analysis of plasma by nested RT-PCR (sensitivity, < 40 copies/mL) and PBMC by IFN-γ ELISpot. Immune hierarchy with dominant responses against nonstructural HCV antigens was preserved in patients, for which the persisting HCV RNA sequence matched the pretreatment sequence (A and B). Immune hierarchy changed from nonstructural proteins to predominantly structural HCV antigens in subject 45 (C) whose HCV RNA sequence (genotype 3) differed from the pretreatment HCV sequence (genotype 1a). Patients depicted in panels A and C had HCV RNA detectable only in the nested round of an RT-PCR, whereas patient 75 (B) had high-level HCV recurrence (see legend to Table 3 for details). (D) Immune responses of patients who tested HCV RNA negative at all studied time points.