In Vivo Determinants of Hepatitis C Virus Adaptation and Escape From Neutralizing Antibody AR5A

Abstract

Background & aims: Mechanisms of hepatitis C virus (HCV) adaptation and escape from broadly neutralizing antibodies (bNAbs) have been primarily studied in vitro. Here, we used a previously developed in vivo adapted J6/JFH1_A876P virus and the highly bNAb sensitive hypervariable region 1 (HVR1) deleted variant, J6/JFH1_A876P,ΔHVR1, to study adaptation and bNAb AR5A escape in the HCV-permissive human-liver chimeric mouse model.

Methods: In vitro identified AR5A escape substitution, L665S, was introduced into J6/JFH1_A876P with or without HVR1. The infection of human liver chimeric mice with these recombinants revealed adaptive mutations, and the potential mechanism of adaptation was extensively characterized in vitro. Finally, we tested the barrier to resistance of AR5A in vivo by challenging passively immunized animals with HVR1-deleted viruses, either with or without the AR5A escape substitution, L665S.

Results: L665S was found to be an escape substitution in vivo. Furthermore, sequence analysis showed that the escape substitution L665S arose as early as 2 weeks post infection. At week 8, we also identified antibody escape substitutions as well as several potential in vivo adaptive substitutions in E2. For J6/JFH1_A876P, S449P and M702L increased cell-free particle infection and broadly affected antibody sensitivity for virus with HVR1. For J6/JFH1_A876P,ΔHVR1, N430D and M702L substitutions increased both cell-free particle mediated infection and cell-to-cell spread, whereas N430D also increased thermal stability at 37°C.

Conclusions: We show that L665S is an AR5A escape mutation in vivo, supporting the use of cost-effective vaccine escape studies in vitro. We also identify novel in vivo adaptive mutations and characterize their mechanism of action, thus facilitating interpretation of future HCV in vivo studies.

Keywords: AR5A; Antibody Escape; Hepatitis C Virus; Human-liver Chimeric Mice; In Vivo Adaptation.

Graphical abstract

Figure 1

Infection of human-liver chimeric mice with HCV recombinants harboring the AR5A in vitro escape substitutions S680T/L665S. (A) Huh 7.5 cells were transfected with the in vitro transcribed RNA of indicated recombinant viruses. The values in the graph represent the HCV titers collected after 72 hours post-transfection as the mean of 4 replicates ± SEM. (B) First passage stocks of the respective recombinant viruses were subjected to 4 replicates of 5-fold dilution from 250 to 0.038 μg/mL of AR5A for virus harboring HVR1 or 10-fold dilution from 50 to 5 × 10^-5 μg/mL of AR5A for HVR1-deleted viruses. The values in the graphs represent the IC₅₀ values, and the error bars represent SD. (C) Human-liver chimeric mice were infected with the culture-derived HCV recombinants. (D) J6/JFH1_A876P/S680T and (E) J6/JFH1_{ΔHVR1/A876P/S680T} with or without L665S (A876P is a previously described in vivo adaptive substitution in NS2). The graphs in (DandE) show the HCV RNA titers from individual animals of a total of 3 mice per recombinant virus monitored in plasma until 16 weeks post-infection. HCV RNA was further extracted from plasma collected during weeks 2 and 8, and the sequences of the amplified HCV Core-NS2 region was determined (Table 1 only for week 8). ^†Mouse was sacrificed because humane endpoints were met.

Figure 2

Effect of in vivo derived substitutions N430D or M702L with or without known AR5A escape substitutions in virus infectivity, antibody susceptibility, and particle stability in 37°C in the HCV recombinant J6/JFH1_ΔHVR1/A876P. (A) Huh7.5 cells were transfected with in vitro-transcribed RNA of the respective HCV recombinants. The values in the graph represents the HCV titers in collected supernatant after 72 hours post-transfection as the mean of 4 replicates ± SEM. (B–C) Huh7.5 cells were transfected with RNA, and transfected cells were mixed with naive cells and plated in 96-well plates and treated with 50 μg/mL C211 polyclonal antibody (B) or 10 μg/mL of AR3A (C) to avoid cell-free particle infection. The values represent the fold increase of single spots per FFU in 24 hours calculated in 6 replicates per each condition using the BioSpot software (Cellular technology Lmtd) following HCV-specific staining at 24- and 48-hours post-transfection. Error bar represents ± SEM. (D–E) First passage stocks were generated, and the indicated HCV recombinants were subjected to 4 replicates of 10-fold dilution series from 50 to 5 × 10^-5 μg/mL of AR5A (D) or AR3A (E). The values in the graphs represent the IC₅₀ values and the error bars represent SD. (F) The temperature stability was tested by incubating the viruses at 37°C for 0.5, 1, 2, 3, 4, 6, and 8 hours prior to infection in Huh7.5 cells, and the half-life was calculated for each specific recombinant. (G–I) Huh7.5 cells were incubated with a dilution series of an antibody that block the interaction of HCV with cellular receptor CD81 (G), SR-BI (H), and LDLr (I) in quadruplicates with 8 replicates of only medium prior to infection with the indicated recombinants. Values in the graph represent the percentage of infectivity as the mean of 4 replicates ± SEM. The data were analyzed by using 1-phase exponential decay curve fitting. Statistical analysis was carried out by 1-way ANOVA or 2-way ANOVA, significance is indicated as: ∗P < .05; ∗∗P < .005; ∗∗∗P < .0005; and ∗∗∗∗P < .0001.

Figure 3

Effect of in vivo derived substitutions or M405V, S449P, and M702L with or without known AR5A escape substitutions in virus infectivity, antibody susceptibility, particle stability in 37°C, and SR-BI dependency in the HCV recombinant J6/JFH1_A876P. (A) Huh7.5 cells were transfected with in vitro transcribed RNA of the respective HCV recombinants. The values in the graph represents the HCV titers in collected supernatant after 72 hours post-transfection as the mean of 4 replicates ± SEM. (B–C) Huh7.5 cells were transfected with RNA and transfected cells were mixed with naive cells and plated in 96-well plates and treated with 1000 μg/mL C211 polyclonal antibody (B) or 250 μg/mL of AR3A (C) to avoid cell-free particle infection. The values represent the fold increase of single spots per FFU in 24 hours calculated in 6 replicates per each condition using the BioSpot software (Cellular Technology Lmtd) following HCV-specific staining at 24- and 48-hours post-transfection. Error bar represents ± SEM. (D–E) First passage stocks were generated, and the indicated HCV recombinants were subjected to 4 replicates of 10-fold dilution series from 100 to 2.5 × 10^-4 μg/mL of AR5A (D) or AR3A (E). The values in the graphs represent the IC₅₀ values, and the error bars represent SD. (F) The temperature stability was tested by incubating the viruses at 37°C for 0.5, 1, 2, 3, 4, 6, and 8 hours prior to infection in Huh7.5 cells, and the half-life was calculated for each recombinant. Values in the graph represent the percentage of infectivity as the mean of 4 replicates ± SEM. The data were analyzed by using 1-phase exponential decay curve fitting. (G–I) Huh7.5 cells were incubated with a dilution series of an antibody that block the interaction of HCV with cellular receptors CD81 (G), SR-BI (H), and LDLr (I) in quadruplicates with 8 replicates of only medium prior to infection with the indicated recombinants. The values in the graph represent the percentage of HCV infection inhibition reached at 12.5 μg/mL of antibody ± SEM. Statistical analysis was carried out by 1-way ANOVA or 2-way ANOVA; significance is indicated as: ∗P < .05; ∗∗P < .005; ∗∗∗P < .0005; and ∗∗∗∗P < .0001.

Figure 4

AR5A protection studies of human-liver chimeric mice against HCV. (A) Human-liver chimeric mice were inoculated intraperitoneally with 0.2 mg of AR5A before being challenged intrasplenically with the indicated HVR1-deleted recombinant, and HCV RNA titers were monitored in plasma until 17 weeks post-challenge. HCV RNA was further extracted from plasma during weeks 2 and 8, and the sequence of the HCV core-NS2 sequence was determined (Table 2 and 3). (B) AR5A inoculation protected animals against infection with virus without L665S; however, virus escape was observed in 2 animals. Sequence analysis of recovered viruses showed that they acquired L665S as early as week 2 postinfection. (C) Virus harboring the escape substitution, L665S, were able to replicate in the presence of AR5A. Dashed line indicates the assay detection limit. ^†Mouse was sacrificed because humane endpoints were met.