Figure 1.
Cell culture model systems for analysis of HCV dissemination and cell-cell transmission.
(A) Transmission pathways of HCV. HCV can disseminate through two routes of transmission: cell-free entry and cell-cell transmission. Cell-free entry is the classical pathway for initiation of HCV infection and requires a well-defined panel of host entry factors including SR-BI, CD81, CLDN1, OCLN and the EGFR signaling pathway [6]-[10], [12]. Cell-free entry does not require cell-cell contact and is efficiently inhibited by neutralizing antibodies (nAbs). Cell-cell transmission requires direct cell-cell contact and is resistant to nAbs present in HCV-infected patients. Cell-cell transmission is considered to play a key role for viral spread and maintenance of infection [4], [31]. (B) HCV spread and dissemination assay. The HCV spread assay monitors viral spread in cell cultures using a low number of Huh7.5.1 cells containing replicating HCVcc, which expand over a time period of 14 days [6], [8]. In the absence of nAb or presence of control antibody the virus is transmitted by cell-cell and cell-free spread (upper panel [6], [8]). Cell-free transmission can be blocked by anti-HCV nAbs allowing to study viral spread mediated by cell-cell transmission only (lower panel). (C) HCV cell-cell transmission assay [6], [7], [11]. The cell-cell transmission assay allows the investigation of cell-cell transmission and its inhibition by HTEIs. In this assay HCV+ Huh7.5.1 producer cells containing replicating HCVcc (stained by an anti-NS5A antibody and indicated in red) are co-cultured with HCV− Huh7.5 GFP target cells (indicated in green) for 24 h in the presence of nAb blocking cell-free transmission. Flow cytometry is used to quantitate infected HCV+ Huh7.5 GFP target cells (indicated by red and green), which are infected via cell-cell transmission and stained with anti-NS5A antibody.
Figure 2.
The spread of DAA-resistant viruses is resistant to neutralizing antibodies against HCV but is efficiently inhibited by HTEIs.
(A–F) DAA-resistant variant (Luc-Jc1 A156S in (B), (D) and (F)) or wild-type HCVcc (Luc-Jc1 in (A), (C) and (E)) transfected Huh7.5.1 cells and uninfected Huh7.5.1 cells were incubated in the presence of isotype control antibody (mouse IgG, 25 µg/mL) (total transmission) (A–B), anti-HCV neutralizing antibody (anti-E2 mAb, AP33, 25 µg/mL) to block cell-free transmission (cell-cell transmission) (A–B), 1 µM of telaprevir (C–D), 10 µg/mL of anti-CLDN1 mAb (E–F) or 10 µM of erlotinib (E–F) 2-3 days after transfection. Different media were replenished every 3–4 days. HCV infection was measured by luciferase reporter gene expression in cell lysates at indicated time points as described [36]. (G) HCVcc (Luc-Jc1) was preincubated with cell culture medium (mock control) or control IgG (25 µg/ml) or nAb (AP33 or anti-HCV IgG, 25 µg/ml) for 1 h and then incubated with Huh7.5.1 cells for 4 h at 37°C. HCV load was analyzed 72 h post-infection by luciferase activity. (H) CD81−Huh7.5 cells or Huh7.5 cells were transfected with HCV Luc-Jc1 and maintained in cell culture over 2 weeks. The intracellular viral load was monitored by measuring luciferase activity every 2–4 days. Means ± SD from at least three independent experiments performed in triplicate are shown.
Figure 3.
Cell-cell transmission is the main transmission route for DAA-resistant viruses.
The spread assay was performed as shown in Figure 2 with or without 25 µg/mL anti-HCV IgG to neutralize cell-free transmission of virus. The relative percentage HCV-positive cells/total cells was determined by immunostaining for NS5A and flow cytometry. Huh7.5.1 uninfected cells were used as a negative control (“uninfected”). (A–B) Percentage of wild-type (WT) (A) or DAA-resistant HCV (A156S) (B)-infected cells without treatment (mock) or in the presence of 25 µg/mL anti-HCV IgG (nAb), 10 µg/mL anti-CLDN1 mAb or 1 µM telaprevir treatment as described in Figure 2 is shown. (C) The supernatant (SN) with or without anti-HCV IgG containing cell-free wild-type or A156S HCV was used to infect fresh Huh7.5.1 cells. The cell culture medium was taken as a negative control. The data are represented of three experiments.
Table 1.
The CLDN1-specific antibody is efficient in inhibiting HCV spread.
Figure 4.
HTEIs effectively block cell-cell transmission of DAA-resistant viruses.
The experimental setup is shown in Figure 1C. NS5A+ HCV producer cells (Pi) were transfected with HCV RNA encoding for HCV Jc1 NS3-A156S (A-B) or Jc1 NS3-L36M/R155K (C–D). NS5A+ HCV producer cells and GFP-expressing target cells (T) were co-cultivated with nAb (anti-HCV IgG, 25 µg/mL) to block cell-free transmission as described [6]. Cell-cell transmission of wild-type or drug-resistant strains was determined by quantification of GFP+ NS5A+ target cells (Ti) by flow cytometry. Protease or NS5A inhibitor-resistant HCV variant producer cells (Pi) cultured with uninfected target cells (T) were then incubated with 1 µg/mL of CLDN1-specific mAb or 10 µM of erlotinib or control medium. HCV-infected target cells (GFP+NS5A+) were quantified by flow cytometry (A and C). Percentage of infected target cells is shown as histograms (B and D) and is represented as means ± SD from three experiments performed in triplicate. *p<0.005.
Figure 5.
Addition of HTEIs to DAA prevents the emergence of DAA-resistant variants.
(A) Huh7.5.1 cells were transfected with RNA encoding wild-type HCV Jc1 and plated in the presence of 1% DMSO and treated with anti-CLDN1 mAb (10 µg/mL), simeprevir (500 nM) alone or in combination with anti-CLDN1 mAb (10 µg/mL) or in the absence of treatment (CTRL). The combination treatment was stopped on day 51 while anti-CLDN1 mAb and simeprevir in monotherapy continued until day 58. Viral load was assessed by RT-PCR every 3–4 days. The limit of quantification (LOQ), indicated by a dashed line, was 103 copies/mL. The experiment was performed in triplicate and repeated twice. Among the detected triplicate samples, one out of three was HCV RNA negative on day 27 (empty circle under the LOQ), two out of three negative on day 30 (empty circles under the LOQ), three out of three negative from day 37 on. The undetectable HCV load from day 40 was confirmed by a clinically licensed HCV RNA detection assay, the Abbott RealTime HCV assay (Abbott), and is indicated by a star (LOD of Abbott qRT-PCR is 48 IU/mL with 250 µL liquid sample). (B) A similarly designed experiment was performed using anti-SR-BI mAb NK-8H5-E3 instead of anti-CLDN1 mAb. (C) Combination of daclatasvir and simeprevir fails to clear HCV genotype 2 infection. Using the same assay as described above, the cells were treated with 5 nM daclatasvir, 500 nM simeprevir, combination of 5 nM daclatasvir and 500 nM simeprevir or mock (CTRL). Viral load was assessed by RT-PCR every 3–4 days. Means ± SD from a representative experiment performed in triplicate are shown.
Table 2.
Absent toxicity in Huh7.5.1 cells treated with an HTEI and/or a DAA or 2 DAAs.
Figure 6.
Analysis of DAA-resistant mutations emerged during treatment protocols by direct sequencing.
In the long-term HCV infection assay shown in Figure 5A, HCV RNA in the supernatants from different treatments was purified on day 23. Direct sequencing was performed to identify predominant viral mutations in HCV NS3 protease region as described in Materials and Methods. Data are displayed as NS3 amino acid sequence in treated cells.
Figure 7.
Mutational analysis of viral variants with treatment of protease inhibitors or/and HTEIs in the long-term HCV infection assay.
Clonal sequencing of HCV NS3 mutations in simeprevir monotherapy and the combination of simeprevir and anti-CLDN1 mAb. To further identify simeprevir induced DAA-resistance mutations, HCV RNA from (A) mock (CTRL), (B) 10 µg/mL anti-CLDN1 mAb, (C) 500 nM simeprevir or (D) 500 nM simeprevir +10 µg/mL anti-CLDN1 mAb treatment on day 23 as shown in Figure 5A and 6 was isolated and amplified as described in Materials and Methods. Following cloning and sequencing of the NS3 protease region, the relative distribution of viral variants (wild-type (WT) and NS3 mutations listed in a clockwise order beside the pie charts) was analyzed and is indicated in different shades of grey in the pie charts. The major DAA-resistance mutation, D168V, is highlighted in blue and WT is in light grey. For each variant, the number of detected clones is indicated in the parenthesis.
Table 3.
Analysis of NS3A and NS5A mutations during DAA monotherapy or treatment with a combination of DAAs.