Recombinant Human Anti-HCV E2 Antibody (HC84-1) (CAT#: PABL-120)

Figure 1 Crystal structure of the epitope II peptide from the complex with HC84-1 (A) and the complex with HC84-27 (D) is shown as cartoon displaying the side chains as sticks. View on the side of the peptide facing the paratope. Residues W437-F442 make a 1.5 α-helical turn followed by an extended segment containing residues Y443-K446.

Krey, T., Meola, A., Keck, Z. Y., Damier-Piolle, L., Foung, S. K., & Rey, F. A. (2013). Structural basis of HCV neutralization by human monoclonal antibodies resistant to viral neutralization escape. PLoS pathogens, 9(5), e1003364.

Figure 2 (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.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J.,... & Bukh, J. (2012). Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.PLoS pathogens, 8(4), e1002653.

Figure 3 (D) Dose-dependent neutralization of JFH1-based genotypes 1–5 C-NS2 recombinant viruses by HMAb HC-84.1 were determined by FFU reduction assay. IC50 values for each respective antibody against different genotype HCVcc are as indicated.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J.,... & Bukh, J. (2012). Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.PLoS pathogens, 8(4), e1002653.

Figure 4 (A) Antibody binding to H77C (wt) and D535A recombinant E2 lysates by ELISA. The assays were performed with 1 µg of E2/ml that was captured by GNA pre-coated wells, and followed by incubation with each HMAb at 1 µg/ml (x-axis). Positive control is HC-1, an antigenic domain B HMAb and negative control is R04. The y-axis shows the mean optical density (O.D.) values.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J.,... & Bukh, J. (2012). Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.PLoS pathogens, 8(4), e1002653.

Figure 5 (B) Immunoprecipitation of 1a H77C recombinant E1E2 lysate by each HCV-84 HMAb (as indicated at the top of the panel). HC-1, an antigenic domain B HMAb, was used as a positive control and R04 was used as a negative control. The immunoprecipitated pellet was separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis under reducing conditions, and immunoblots were analyzed with HMAbs recognizing linear epitopes: anti-E2, HC-33.1 and anti-E1, H-111.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J.,... & Bukh, J. (2012). Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.PLoS pathogens, 8(4), e1002653.

Figure 6 (C) HC-84 HMAbs do not bind to denatured 1a HCV E2. Recombinant E1E2 lysate was either left untreated (black bars) or denatured by incubation with 0.5% sodium dodecyl sulfate and 5 mM dithiothreitol for 15 min at 56°C (red bars). After treatment, the proteins were diluted 1∶5 in BLOTTO and captured by pre-coated GNA wells. After washing and blocking, bound proteins were incubated with each HC-84 HMAb at 5 µg/ml (x-axis) and a control HMAb, HC-33.1. Bound antibody was detected as described in Materials and Methods. The y-axis shows the mean optical density values for triplicate wells.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J.,... & Bukh, J. (2012). Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.PLoS pathogens, 8(4), e1002653.

Figure 7 (A) Dual antibody immunofluorescence staining of Huh7.5 cells infected with JFH1 2a virus after multiple rounds of neutralization by the respective antibody. R04, a human monoclonal antibody against CMV was used as mock selection. HCV E2 glycoprotein was stained with the respective antibody under which viral escape mutants were selected (green). Total virus infected cells were stained with anti-NS3 antibody labeled with Alexa-594 (red). The cells were counterstained with Hoechst nuclear stain H33342 (blue). Escape mutants were assessed for CBH-2 (a neutralizing domain B HMAb [43]) at passage 3 in 10 µg/ml, HC-84.1 at passage 4 in 0.5 µg/ml

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J.,... & Bukh, J. (2012). Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.PLoS pathogens, 8(4), e1002653.