Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients

Abstract

Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.

FIG. 1.

Sequence analysis of the variable domains of HCV-AB 68 and HCV-AB 65. RNA was amplified by RT-PCR, cloned, and sequenced as described in Materials and Methods. (A) V_L (light chain) amino acid sequences of HCV-AB 68 and HCV-AB 65. (B) V_H (heavy chain) amino acid sequences of HCV-AB 68 and HCV-AB 65. Complementarity-determining regions are in boldface type.

FIG. 2.

Competition analysis between HCV-AB 68 and HCV-AB 65 by the Pair Wise Binding method. BiaCore sensor chip CM5 coated by dextran matrix was bound by amine coupling with E2-CHO, and then the first antibody was injected several times over the coated sensor chip to saturate specific binding sites. The second antibody was then injected, and activity changes measured as resonance units (RU) were monitored. Injections of the antibodies are marked by arrows.

FIG. 3.

Reactivity of HCV-AB 68 and HCV-AB 65 to E2 constructs by Western blot analysis. SDS-PAGE of different E2 constructs under nonreducing and reducing conditions was performed as described in Materials and Methods. (A) Samples prepared at 37°C. (B) Samples prepared at 100°C. Lanes: 1, His-EK-E2; 2, His-EK-E2-without HVR1; 3, E2 MCS; 4, E2 MCS without HVR1; 5, E2-CHO.

FIG. 4.

Immunohistostaining of HCV-infected liver with HCV-AB 68 and HCV-AB 65. Immunohistostaining of human liver tissue with HCV-AB 68 and with HCV-AB 65 was performed on liver fragments from HCV-infected patients as described in Materials and Methods. Liver tissues from HBV-infected patients who were negative for all HCV markers served as a negative control. (A) HCV-AB 68 with HCV-infected liver (magnification, ×40). (B) HCV-AB 68 with HBV-infected liver (magnification, ×40). (C) HCV-AB 65 with HCV-infected liver (magnification, ×40). (D) HCV-AB 65 with HBV-infected liver (magnification, ×40).

FIG. 5.

Immunoprecipitation of virus particles from HCV-infected sera from different genotypes by HCV-AB 68 and HCV-AB 65. HCV particles from infected patients' sera from genotypes 1b, 2a/2c, and 3a were captured by magnetic beads coated with a specific antibody, as described in Materials and Methods. Following magnetic separation, HCV RNA was detected in the bound fraction by RT-PCR. (A) Immunoprecipitation by HCV-AB 68 (in quadruplicates) and HBV-AB 17 as an isotype control (in duplicates). (B) Immunoprecipitation by HCV-AB 65 (in triplicates) and HBV-AB 17 as an isotype control (in triplicates).

FIG. 6.

Complement fixation by HCV-AB 68 and HCV-AB 65 in HCV-infected sera. Complement fixation in HCV-infected patients' sera by HCV-AB 68 and HCV AB 65 was tested as described in Materials and Methods. Serum 1, HBV-infected control; sera 2 to 9, HCV infected. The percent increase in complement fixation was calculated as follows: percent increase in complement fixation = {100 − [percent free complement (with HumAb) × 100]/[percent free complement (without HumAb)]}.

FIG. 7.

Immune complex (IC) formation by HCV-AB 68 and HCV-AB 65 in HCV-infected sera. Formation of IgG IC in HCV-infected patients' sera by HCV-AB 68 and HCV AB 65 was tested as described in Materials and Methods. Serum 1, HBV-infected control; sera 2 to 7, HCV infected. Percent IgG IC was calculated as follows: percent IgG IC = {[OD₄₅₀ (sample) × 100]/[OD₄₅₀ (no HumAb)] − 100}.

FIG. 8.

Complement-dependent phagocytosis of HCV IC formed by HCV-AB 68 and HCV-AB 65. Complement-dependent phagocytosis of IC by neutrophils was tested using fluorescein-labeled HCV-AB 68 or HCV-AB 65 and sera of HCV-infected patients, as described in Materials and Methods. DAPI stains cell nuclei blue, whereas IC are stained green with fluorescein. (A) HCV-AB 68. (B) HCV-AB 65. Panels 1, HCV-infected serum with complement; panels 2, HCV-infected serum with no complement; panels 3: normal human serum.

FIG. 9.

Treatment of HCV-infected Trimera mice by HCV-AB 68 and HCV-AB 65. Two intraperitoneal injections of either HCV-AB 68 or HCV-AB 65 were administered to Trimera mice (n = 17/group) with established HCV viremia on days 16 and 17 post-transplantation (20 μg/mouse/day; a total of 40 μg of antibody per mouse). HCV RNA was measured in mouse sera sampled 1 and 5 days after treatment completion (days 18 and 22). The solid line indicates the limit of detection of the assay, which is 5 × 10³ RNA copies/ml.

FIG. 10.

Inhibition of HCV infection in the HCV Trimera model by a 1:1 (mg/mg) mixture of HCV-AB 68 and HCV-AB 65. Samples of human serum containing 7.5 × 10⁵ HCV RNA copies/ml were preincubated with 100 to 400 μg/ml of a 1:1 (mg/mg) mixture of HCV-AB 68 and HCV-AB 65 and subsequently used to infect normal human liver fragments ex vivo. Following infection, the liver fragments were transplanted in mice (n = 17/group), and HCV RNA was measured in mouse sera 19 days later. The solid line indicates the limit of detection of the assay, which is 5 × 10³ RNA copies/ml.

FIG. 11.

Neutralization of HCVpp genotype 1b infection of Huh-7 cells by HCV-AB 68 and HCV-AB 65. Infection medium containing concentrated HCVpp was preincubated with 20 μg/ml of either HCV-AB 68, HCV-AB 65, or HBV-AB 17 as an isotype control prior to addition to Huh-7 cells as described in Materials and Methods. The virus neutralization activity of an antibody was determined by the percent reduction of luciferase activity compared with the infection medium containing PBS. Experiments were performed in triplicate.