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Correspondence: Hendrik W. Reesink, MD, PhD, Department of Hepatology, Academic Medical Center, PO Box 22660, 1100 DD Amsterdam, The Netherlands. E-mail: firstname.lastname@example.org
Achievement of a sustained virologic response (SVR) after peginterferon (PEG-IFN) and ribavirin (RBV) treatment is considered to be a marker for the cure of chronic hepatitis C virus (HCV) infection. Long-term follow-up of patients with SVR after treatment with a direct acting antiviral has not yet been described. We used a randomized placebo-controlled, double-blind, two-period phase 1b trial that was conducted in 40 HCV genotype 1 (treatment-naïve and treatment-experienced)-infected patients. Nineteen patients achieved SVR after treatment with the HCV protease inhibitor narlaprevir followed by PEG-IFN/RBV. In these patients, HCV-RNA tests were scheduled at 3, 6, 12 and 24 months after end of treatment. Patients were followed for a median of 27 months (range 15–32) after end of treatment with a median number of follow-up visits of 4 (range 3–8). All patients remained HCV-RNA negative over time. SVR achieved following narlaprevir and PEG-IFN/RBV-therapy was durable up to 32 months after the end of treatment.
The standard of care antiviral treatment (AVT) for chronic hepatitis C virus (HCV) infection, peginterferon (PEG-IFN) and ribavirin (RBV) has been modified to include a direct acting antiviral (DAA), either telaprevir or boceprevir . Recently, boceprevir and telaprevir [direct acting antiviral agents (DAA)] have been added to peginterferon (PEG-IFN) and ribavirin (RBV) in the treatment of chronic hepatitis C .
Successful HCV treatment has been defined as achievement of a sustained virologic response (SVR), that is undetectable HCV-RNA 24 weeks after the end of treatment (EoT). SVR has been shown to be durable up to 18 years post-treatment and is therefore considered a marker for successful HCV antiviral therapy . Clinical studies investigating triple therapy regimens have used the same end-point. To date, undetectable HCV-RNA 24 weeks after DAA-based therapy has not yet been described to be a marker of long-term sustained viral eradication. In contrast to PEG-IFN/RBV, DAAs select for resistant HCV strains and their immune modifying capacities are limited [3-6]. Trace amounts of HCV-RNA have been detected in different compartments of the human body years after SVR following PEG-IFN/RBV [7, 8]. Veerapu et al. showed that, with long-term follow-up after achieving SVR, HCV-RNA reappearances in plasma induced HCV-specific T-cell responses . It is not known if traces of HCV-RNA also persist after successful DAA-based therapy, and whether these traces are subject to immune control or could result in a late virologic relapse.
We studied durability of SVR after treatment with the protease inhibitor narlaprevir and PEG-IFN with narlaprevir, followed by PEG-IFN/RBV for 24–48 weeks .
Methods and Materials
We used a randomized, placebo-controlled, double-blind, two-period phase 1b trial that was conducted in 40 HCV genotype (GT) 1-infected (naïve and treatment-experienced) patients . Narlaprevir was administered for 7 days as 800 mg thrice daily without ritonavir or 400 mg twice daily with 200 mg ritonavir twice daily. After a 4-week washout, the same dose and regimen of narlaprevir was administered in combination with PEG-IFN-α-2b for 14 days. Thereafter, all patients initiated PEG-IFN-α-2b and RBV treatment for 24 weeks if a rapid virologic response was achieved, otherwise they received 48 weeks of treatment. Six of 20 (30%) treatment-experienced and 13 of 20 (65%) treatment-naïve patients achieved SVR and these were all randomized to narlaprevir . Patients were followed up at 3, 6, 12 and 24 months after EoT. At each follow-up visit, blood was collected and tested for HCV-RNA using Roche Ampliprep/Cobas Taqman HCV/HPS assay (Roche Molecular Systems Inc., Branchburg, NJ, USA) with lower limit of detection (LLOD) of 15 IU/mL or the VERSANT® TMA HCV-RNA qualitative assay (Siemens Medical Solutions Diagnostics, Eindhoven, the Netherlands) with LLOD of 5.3 IU/mL. At the last follow-up visit, which was at least 24 months after EoT, the VERSANT® TMA HCV-RNA qualitative assay was used.
At the final follow-up visit, in vitro HCV-specific T-cell proliferation and HCV-RNA levels were determined in peripheral blood mononuclear cells (PBMC). Fresh PBMC were cultured in quadruplicate wells of a 96-well round-bottom plate (2 × 105 cells in 200 μL) and stimulated with overlapping peptide pools (1 μg/mL) spanning the core, NS3, NS4, NS5a and NS5b HCV genome (clone J4, GT 1b; BEI Resources, Manassas, VA, USA), CMV antigens (34 μg/mL; AD-169; Microbix, Toronto, Canada) or no stimulus. RPMI 1640 medium supplemented with 5% human serum (Lonza, Verviers, Belgium) and anti-CD28 and anti-CD49d antibodies (both 1 μg/mL; eBioscience, San Diego, CA, USA) were used. At day 5, cells were pulsed for 16 h with [3H]-thymidine (0.5 μCi/well; Amersham, Little Chalfont, UK). Proliferation was determined as counts per minute by liquid scintillation. For HCV-RNA determination in PBMC, cells were lysed by repeated freeze-thaw cycles and tested for HCV-RNA using the VERSANT® TMA HCV-RNA qualitative assay. A parallel study was conducted in 22 treatment-naïve chronic HCV-infected patients treated with PEG-IFN-alfa2b and RBV for 24 weeks (GT 2 and 3) or 48 weeks (GT 1 and 4) . Six of these patients achieved SVR. The results of identical immunologic assays performed with cells from these six patients were compared with those from the patients receiving narlaprevir treatment.
Of the 19 patients who achieved SVR with the narlaprevir-based regimen, 14 (75%) were men, 15 (79%) were of Caucasian race, median age was 56 years (range 33–65 years) and six patients (32%) were previously treated with an IFN-based regimen. At follow-up, three patients (16%) had signs of cirrhosis which was already present before narlaprevir treatment, and median alanine transaminase (ALT) was 23 (range: 11–89). Only the three cirrhotic patients had ALT levels above the upper limit of normal. After EoT, the median number of follow-up visits was 4 (range 3–8) and the median follow-up time in months was 27 (range 15–32). To be more specific regarding patients with a higher chance of relapse, number of follow-up visits and median follow-up time in patients with cirrhosis was 3 and 27 (range 24–30) months, respectively. One patient did not respond to the call for his final visit; his final follow-up visit was 12 months after EoT. All patients had undetectable HCV-RNA at all follow-up visits. In addition, no clinically relevant abnormalities were observed for any patient during follow-up.
In vitro quantification of HCV-specific T-cell proliferation in blood was performed in six patients (one patient with cirrhosis) with SVR after narlaprevir-based therapy at the final follow-up visit, which was at least 24 months after EoT (median 25 months, range 23–30 months). HCV-specific T-cell proliferation was low compared with the positive control CMV and comparable with the negative control of culture medium alone. Also, the six patients who achieved SVR after PEG-IFN/RBV-therapy showed no HCV-specific T-cell proliferation at follow-up when compared to medium (Figs 1a,b). Regarding PBMC, five of six patients tested HCV-RNA negative; the remaining patient tested positive for HCV-RNA. This patient did not have cirrhosis and/or changes in ALT.
In the present long-term follow-up study in HCV GT-1-infected patients, we demonstrated durability of SVR antiviral treatment with the protease inhibitor narlaprevir (+/− ritonavir) followed by narlaprevir/PEG-IFN-2b (+/− ritonavir) and 24–48 weeks of PEG-IFN/RBV. In addition, no detectable HCV-specific T-cell responses were measured in blood, as expected in the absence of HCV-RNA in the circulation.
Sustained virologic response is considered a marker for cure because of its durability and associated improved clinical outcomes [2, 11, 12]. Recently, the protease inhibitors telaprevir/boceprevir have been added to standard of care AVT. Therefore, knowledge of durability and reliability of SVR as surrogate marker with these new regimens is of major importance.
In our study, no reappearances of HCV-RNA during follow-up were observed, and, at final follow-up, T-cell proliferation against HCV in blood was low. In a subset of patients who achieved SVR and were only treated with PEG-IFN/RBV, HCV-specific T-cell proliferation was low as well. These T-cell proliferation findings are in line with the inability to detect HCV-RNA at follow-up; both findings are indicative of viral eradication. These findings are important because in some studies, in which HCV patients achieved SVR with PEG-IFN/RBV, minute amounts of HCV-RNA were detected in the circulation at long-term follow-up [7, 8]. Veerapu et al. demonstrated HCV-specific T-cell responses with detection of small amounts of HCV-RNA . Consequently, the absence of HCV-specific T-cell response supports the undetectable HCV-RNA test results over time, suggesting viral eradication. Because we used a highly sensitive test to detect HCV-RNA (LLOD 5.3 IU/mL), performed frequent and long-term follow-up visits and because a virologic relapse would be expected to result in persistent viremia, we consider SVR durable in these narlaprevir-treated patients. In 1 of 6 patients, PBMC tested positive for HCV-RNA, while other tests, HCV-RNA in the peripheral circulation, HCV-specific T-cell responses and ALT, were not suggestive of viral relapse. With long-term follow-up of patients that achieved SVR after PEG-IFN/RBV, HCV-RNA has been demonstrated in PBMC as well, even in the absence of viral relapse . Our study is limited by the small number of patients. Recently, similar results have been presented in a larger cohort of telaprevir-treated patients, although this has not been published yet . With the introduction of DAAs to PEG-IFN/RBV as standard of care for HCV infection, patients should be monitored long-term until our important clinical findings are validated in larger cohorts and in cohorts treated with other DAA-based regimens.
In conclusion, SVR achieved with narlaprevir in combination with PEG-IFN and RBV was durable, suggesting SVR to be a valid surrogate marker for successful DAA-based treatment for chronic HCV infection.
Conflict of Interest
Michelle A. Treitel is an employee of Merck Research Laboratories. The other authors have no conflicts of interest.
This manuscript was supported by the Foundation for Liver Research, Rotterdam, the Netherlands.