Schematic illustrations of phage display screening method.

The hepatitis C virus (HCV) E2 protein derived from either strain TH (genotype 1b) or strain JFH-1 (genotype 2a) was allowed to bind to the surface of magnetic beads, and the single chain Fv (scFv) phage library then was added to the coated beads. Specifically bound phages were amplified by infection of E. coli. These steps were repeated for a total of 4 times to enrich for phages with specific binding to the HCV E2 protein. To confirm the specific binding of scFv phages to HCV E2 protein-bound magnetic beads, the gene encoding the corresponding IgG was constructed from phagemid scFv-encoding sequences by PCR cloning. Specific binding of these antibodies to HCV E2 protein was assessed by enzyme immune assay (EIA) recognition and neutralization of HCV pseudoparticle (HCVpp) infection. (TIF)</p

List of primers.

(DOCX)</p

Results of phage display screen.

Results of phage display screen.</p

Dosing with anti-E2 antibody e2d066 does not affect serum concentration of human albumin (h-Alb) in human liver chimeric mice.

(A) Study design of in vivo infection experiment. Human liver-transplanted immunodeficient mice were inoculated with J6/JFH-1 cell culture-generated infectious hepatitis C virus particles (HCVcc) on Days 0, 28, and 42 (inoculated HCV RNA copy numbers were 102, 103, and 103 per mouse respectively; filled triangles). Anti-E2 antibody e2d066 or control human IgG were administered by intraperitoneal injection (800 μg/mouse, i.p.; gray triangles) to the mice on Day -1 (i.e., one day before) and Days 1, 4, 8, 11 after the first (Day-0) challenge with J6/JFH-1 HCVcc. Blood samples of the mice were collected once weekly (open triangle) and processed to obtain serum sample. (B) Human albumin in the blood of the chimeric mice was measured with the Alb-II Kit (Eiken Chemical, Tokyo, Japan). Filled points: control IgG treated mice. Open points: e2d066 treated mice. (TIF)</p

List of antibodies.

(DOCX)</p

Comparison of binding affinity to HCV E2 protein of 3 selected IgG clones.

Binding affinity of obtained anti-E2 antibodies to E2 proteins from (A) TH and (B) J6CF was analyzed by enzyme immune assay. The titers of antibodies were measured by a peroxidase assay kit. Open-circle: e2d066 IgG, open-triangle: e2d073 IgG, open-square: e2d081 IgG, filled-square: HR1-007 IgG (control). (TIF)</p

Neutralizing activity of anti-E2 IgG and scFv antibodies against HCVcc infection.

The neutralization effects of anti-E2 antibodies were assessed using an infection system with chimeric HCVcc, including (A) H77/JFH-1, (B) TH/JFH-1, (C) J6/JFH-1 and (D) S310/JFH-1. A total of 200 focus forming unit of these viruses were mixed with IgG or scFv at the designated concentrations and the mixtures were incubated at room temperature for 30 minutes. The antibody-HCVcc mixture was added to naïve Huh-7.5.1 cells (2 x 104 cells/well, 48-well plate) at 100 μL/well and plates were incubated in a 5% CO2 incubator at 37°C for 3 hours. The antibody-HCVcc mixture then was removed and replaced with 500 μL of DMEM containing 10% FBS, and the cells were cultured in a 5% CO2 incubator at 37°C for a further 72 hours. The cells then were washed with PBS, and Passive Lysis Buffer (Promega) was added at 100 μL/well to generate a cell lysate. HCV core protein in the collected cell lysate was quantified using a Lumipulse Ortho HCV Ag (Ortho Clinical Diagnostics, Tokyo, Japan). The efficiency of neutralization was calculated and presented as the % neutralization normalized to the amount of HCV core protein in the HCVcc-PBS mixture-inoculated cell lysate. Assay were performed in triplicate and infection rate are expressed as mean ± SEM. Statistical significance of difference was analyzed using one-way ANOVA with a Williams’ test (*P < 0.025, **P < 0.005, ***P < 0.0005 vs PBS).</p

Schematic illustration of the method used to isolate anti-E2 antibody neutralization-resistant virus.

Each IgG antibody was diluted with phosphate-buffered saline (PBS) and combined with J6/JFH-1 cell culture-generated infectious hepatitis C virus particles (HCVcc); the mixture was allowed to react at 37°C for 1 hour. The antibody-HCVcc mixture then was added to Huh7 cells seeded in a 12-well plate to permit infection of the cells with HCVcc. Three days after the infection, the cells were subcultured into a 6-well plate, and the culture supernatant was collected at 3 and 6 days after the start of subculturing. An infectious titer measurement was performed to confirm that the collected culture supernatant contained J6/JFH-1 HCVcc. The culture supernatants that contained J6/JFH-1 HCVcc were used for subsequent infection, such that the collected culture supernatant again was mixed with the antibody, and the mixture was added to uninfected Huh7 cells. These steps were repeated for a total of 8 times. The infection-inhibiting activity of the anti-E2 antibody against the virus isolated at the final round (J6/JFH-1_EM) was determined to assess the possible presence of an escape mutant. (TIF)</p

Neutralizing activities of phage library-derived IgGs against HCVpp infection.

The neutralization effects of the e2d066, e2d073, and e2d081 antibodies were assessed by infection assays using HCV pseudoparticles (HCVpp) harboring the E1 and E2 glycoproteins of (A) TH (genotype 1b) and (B) J6CF (genotype 2a). HCVpp, which include a luciferase-encoding construct, were mixed with immunoglobulin G (IgG) or phosphate buffered saline (PBS) for 30 minutes at room temperature; the mixtures then were used to inoculate naïve Huh-7.5.1 cells. Monoclonal anti-CD81 antibody (JS-81, BD Pharmingen) was used as positive control in this assay. At 72 hours after infection, the infected cells were harvested and lysed with Cell Culture Lysis Reagent (Promega). Luciferase activity was quantified using the Luciferase Assay System (Promega). Neutralizing activity was calculated and is presented as the % neutralization by comparison with the luciferase activities of the well inoculated with the HCVpp-PBS mixture. Assay were performed in triplicate and infection rate are expressed as mean ± SEM. Statistical significance of difference was analyzed using one-way ANOVA with a Williams’ test (*P < 0.025, **P < 0.005, ***P < 0.0005 vs PBS).</p

Neutralizing effect of e2d066 against J6/JFH-1 HCVcc infection in human liver chimeric mice.

Neutralizing effect of e2d066 against J6/JFH-1 HCVcc infection in human liver chimeric mice.</p