Figure 2. Breadth of neutralization and inhibition of E2 binding to CD81 by HC-84 HMAbs.

(A) Dose-dependent neutralization of 1a H77 HCVcc and (B) 2a JFH1 HCVcc, as determined by FFU reduction assay [27], [43]. The antibodies are ordered from the lowest to the highest concentration required to reach 50% maximal neutralization concentration (IC₅₀), as summarized in Table 1. Infectious 1a H77C (HJ3–5) chimeric virus or 2a JFH1 inoculum was incubated with each HMAb, at concentrations ranging from 0.005–20 µg/ml against 1a H77C and from 0.0005–20 µg/ml against 2a JFH1, prior to inoculation onto Huh7.5 cells that were pre-seeded in eight-well tissue culture chamber slides. Cells were fixed and immunostained with a MAb to NS3 antigen at day 4 p.i., and enumerated by FFU-reduction assay. Each assay was performed in triplicates and data are shown as percent neutralization, the mean of three experiments ±SD. (C) Neutralization of JFH1-based genotypes 1–6 C-NS2 recombinant viruses as determined by FFU reduction [60]–[62]. The designation of the viruses are: genotype 1a (H77C/JFH1), 2a (J6/JFH1), 3a (S52/JFH1), 4a (ED43/JFH-1), 5a (SA13/JFH1) and 6a (HK6a/JFH1) [11], [60], [63], [64]; all except the 2a virus contained adaptive mutations. R04 is an isotype-matched HMAb negative control. Infectious virus inoculum was incubated with each HMAb at 50 µg/ml followed by inoculation onto Huh7.5 cells. Cells were immunostained with a MAb to NS5A antigen at 45 hrs p.i., and enumerated by FFU. The error bars are SEMs of 8 replicates (from 4 replicates each in 2 independent assays) compared with 12 replicates of virus only (from 6 replicates each in 2 independent assays). (D–E) Dose-dependent neutralization of JFH1-based genotypes 1–5 C-NS2 recombinant viruses by (D) HMAb HC-84.1 and (E) HC-84.26 were determined by FFU reduction assay. IC₅₀ values for each respective antibody against different genotype HCVcc are as indicated. (F) Dose-dependent neutralization of HC-84.1 and HC-84.26 against JFH1-based genotype 6a recombinant virus by FFU reduction assay. R04 is an isotype-matched HMAb negative control. IC₅₀ values for each antibody are as indicated. Infectious virus inoculum was incubated with each HMAb at 0.005–50 µg/ml (in D and E) or 0.00005–50 µg/ml (in F) followed by inoculation onto Huh7.5 cells. Cells were immunostained with a MAb to NS5A antigen at day 2 p.i., and enumerated by FFU. The error bars are SEMs of 4 replicates compared with 6 replicates of virus only. (G) Inhibition of E2 binding to CD81-LEL by HC-84 HMAbs. Genotype 1a H77C recombinant E1E2 lysate containing 1 µg/ml E2 was incubated with each test HMAb at 1 and 10 µg/ml, and the antibody-antigen complex was then added onto CD81-LEL-precoated wells. Detection of E2 bound to CD81-LEL was measured with biotinylated CBH-4D [18]–[20]. HC-11 was used as a positive control. The experiments were performed twice in triplicate. Error bars indicate one standard deviation from the mean.