Recombinant Human Anti-HCV E2 Antibody (HC-84.1)

(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.

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.

(A) HC-84.21 dose-dependent binding, 0.005–2 µg/ml, was measured by ELISA against E2 mutants bearing alanine substitutions at L441, F442 or Y443, and wt H77C. Data are shown as mean optical density (O.D.) values ±SD of two experiments performed in triplicate.

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.

(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.

(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, HC-84.20 at passage 4 in 0.1 µg/ml, HC-84.23 at passage 1 in 10 µg/ml, HC-84.24 at passage 2 in 0.05 µg/ml and HC-84.25 at passage 5 in 0.5 µg/ml. *Only HC-84.1 is shown to represent the tested HC-84 antibodies.

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.