Human Anti-HCV E2 Recombinant Antibody (Clone HC84-26) (CAT#: PABS-0067)

Tabel 1 Binding kinetics of HC84.26 affinity mature clones against 1a H77C E2 and variants

Keck, Z. Y., Wang, Y., Lau, P., Lund, G., Rangarajan, S., Fauvelle, C., ... & Foung, S. K. (2016). Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice. Hepatology, 64(6), 1922-1933.

Tabel 2 Neutralization against 1a H77Cpp and variants

Keck, Z. Y., Wang, Y., Lau, P., Lund, G., Rangarajan, S., Fauvelle, C., ... & Foung, S. K. (2016). Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice. Hepatology, 64(6), 1922-1933.

Figure 1 Human liver-chimeric mice received HC84.26.5D at 250 mg/kg by intraperitoneal injection 24 hours prior to challenge with genotype 1b infected human serum (10 ⁵ IU of HCV RNA) by intravenous injections.

Viral load measurements were determined by realtime PCR at weekly intervals on an ABI model 7300 system using TaqMan chemistry.

Keck, Z. Y., Wang, Y., Lau, P., Lund, G., Rangarajan, S., Fauvelle, C., ... & Foung, S. K. (2016). Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice. Hepatology, 64(6), 1922-1933.

Figure 2 Serum human IgG half-life measurements in mice treated with HC84.26.5D.

Each timepoint was measured by ELISA in triplicates for HMAb in each mouse serum sample tested and the concentration was back-calculated based on a standard curve.

Keck, Z. Y., Wang, Y., Lau, P., Lund, G., Rangarajan, S., Fauvelle, C., ... & Foung, S. K. (2016). Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice. Hepatology, 64(6), 1922-1933.

Figure 3 Unlabeled HC33 HMAbs were used to compete against the corresponding biotinylated HC33 HMAbs to define maximum competition (Self).

Unlabeled HC84 or HC-11 HMAb at 50 μg/ml (x axis) was incubated with E2 that had been immobilized on a GNA-precoated ELISA plate. Biotinylated HC33 HMAbs at 2 μg/ml were then added, and bound biotinylated HMAbs were measured with AP-conjugated streptavidin.

Keck, Z., Wang, W., Wang, Y., Lau, P., Carlsen, T. H., Prentoe, J., ... & Foung, S. K. (2013). Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein. Journal of virology, 87(1), 37-51.

Figure 4 Cooperativity in functional effects of combined HMAbs to aa 412 to 423 and aa 434 to 446.

Average CI values at FAs of 0.5, 0.75, and 0.9 (±SD) for virus neutralization (A) and for blocking E2 binding to CD81 (B) for each HC33 HMAb combined with HC84.20, HC84.26, or HC-11. The dotted lines indicate ranges for antagonistic, additive, or synergistic effects by their respective CI values.

Keck, Z., Wang, W., Wang, Y., Lau, P., Carlsen, T. H., Prentoe, J., ... & Foung, S. K. (2013). Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein. Journal of virology, 87(1), 37-51.

Figure 5 Antigenic characterization of E2 designs using ELISA.

Designs were cloned and expressed in the context of E1E2 as previously described (28) and tested in for binding to a panel of HMAbs that target E2 antigenic domain A (CBH-4G, CBH-4B), B (HC-1), C (CBH-7), D (HC84.28, HC84.24, HC84.26), and E (HC33.1, HC33.4), at concentrations of 1 μg/ml, and 5 μg/ml. Binding was tested to wild-type H77 E2 (sE2; residues 384-661), and compared with sE2 designs ΔHVR1 (residues 408-661), ΔHVR1411 (residues 411-661), H445P, V627NT (V627NV629T), R630NT (R630N-Y632T), K628NS (K628N-R630S), and Y632NS (Y632N-G634S). Asterisks denote designs that were tested in the context of ΔHVR1411 (aa 411-661) sE2 rather than full length sE2 (384-661).

Pierce, B. G., Keck, Z. Y., Wang, R., Lau, P., Garagusi, K., Elkholy, K., ... & Foung, S. K. (2020). Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization. Journal of virology, 94(22), e00704-20.

Figure 6 Measured binding of broadly neutralizing monoclonal antibody HC84.26.WH.5DL to E2 design H445P compared to wild-type soluble E2 (sE2).

Pierce, B. G., Keck, Z. Y., Wang, R., Lau, P., Garagusi, K., Elkholy, K., ... & Foung, S. K. (2020). Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization. Journal of virology, 94(22), e00704-20.

Figure 7 Binding of peptides to control monoclonal antibodies HC33.1, AP33, and HC84.26.WH.5DL.

Pierce, B. G., Keck, Z. Y., Wang, R., Lau, P., Garagusi, K., Elkholy, K., ... & Foung, S. K. (2020). Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization. Journal of virology, 94(22), e00704-20.

Figure 8 Binding of purified HCV pseudoparticles (HCVpps), pseudotyped with H77C E1E2, to monoclonal antibodies.

Binding measurements were performed using ELISA with antibodies targeting E2 (HCV1, HC84.26.WH.5DL, AR3A), E1E2 (AR4A, AR5A) and a negative control antibody (CA45).

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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 9 Dose-dependent neutralization of 1a H77 HCVcc as determined by FFU reduction assay.

The antibodies are ordered from the lowest to the highest concentration required to reach 50% maximal neutralization concentration (IC50). Infectious 1a H77C (HJ3-5) chimeric virus was incubated with each HMAb, at concentrations ranging from 0.005-20 µg/ml against 1a H77C, 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., ... & Foung, S. K. (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 10 Dose-dependent neutralization of 2a JFH1 HCVcc, as determined by FFU reduction assay.

The antibodies are ordered from the lowest to the highest concentration required to reach 50% maximal neutralization concentration (IC50). Infectious 2a JFH1 inoculum was incubated with each HMAb, at concentrations ranging 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., ... & Foung, S. K. (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 11 Neutralization of JFH1-based genotypes 1-6 C-NS2 recombinant viruses as determined by FFU reduction.

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); 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.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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 12 Dose-dependent neutralization of JFH1-based genotypes 1-5 C-NS2 recombinant viruses by HC-84.26 were determined by FFU reduction assay.

IC50 values for each respective antibody against different genotype HCVcc are as indicated. Infectious virus inoculum was incubated with each HMAb at 0.005-50 µg/ml followed by inoculation onto Huh7.5 cells.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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 13 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 0.00005-50 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.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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 14 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. HC-11 was used as a positive control.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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 15 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., ... & Foung, S. K. (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 16 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., ... & Foung, S. K. (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 17 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.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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 18 HC-84 HMAb binding to epitope-II by ELISA.

Biotinylated-epitope II, aa434-446, was captured by streptavidin in microtiter wells. The wells were then incubated with each HC-84 HMAb at 10 µg/ml, serum (1∶100 dilution) from the individual whose B cells were employed to isolate the HC-84 antibodies, and a normal human serum (1∶100 dilution), as a negative control. Specific binding was detected by anti-human IgG-labeled horseradish peroxidase.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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 19 Peptide (epitope-II) inhibition of HC-84 antibody binding to E2.

Recombinant H77C E1E2 lysate was captured by GNA in microtiter wells. The wells were then incubated with selected HC-84 HMAbs and two controls, HC-11 and R04 that were pre-incubated with epitope-II (labeled as peptide) at indicated concentrations. Binding was detected after anti-human IgG-labeled horseradish peroxidase.

Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T., Prentoe, J., ... & Foung, S. K. (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.