Journal: PLoS Pathogens
Article Title: Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents
Figure Lengend Snippet: 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 10 3 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.
Article Snippet: Most interestingly, during combination treatment, HCV RNA became undetectable by qualitative RT-PCR and using the clinically licensed Abbott RealTime HCV assay (Abbott) with a limit of detection of 48 IU/mL ( ).
Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, RNA Detection, Quantitative RT-PCR, Infection