Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody

Abstract

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.

Figure 1. Human CLDN1 expression and tight junction ultrastructure in the livers of human chimeric mice

(a) Human CLDN1 expression in non-infected chimeric livers of uPA-SCID mice (left panel) as well as non-infected human livers (right panel) was assessed by confocal microscopy as described in Methods. Representative 3D composite images show human CLDN1 (green) co-stained with apical membrane marker human CD10 (red). Scale bars – 20 μm. (b) Binding of CLDN1-specific mAb to hepatocyte ultrastructures was assessed by transmission electron microscopy analyses and immunogold labeling of tissue sections of chimeric human mouse livers from mAb-treated mice. Images show TJ area (left panels) or basolateral membranes of hepatocytes (right panels). Red arrows indicate TJ, empty triangles indicate immunogold staining. Scale bars – 500nm. (c) Confocal microscopy of polarized HepG2-CD81 HCV permissive cells stained with CLDN1-specific antibody. HepG2 DsRED-CD81 cells were stained live with CD81- (upper panels) or CLDN1-specific (lower panels) antibodies and visualized by confocal microscopy. CLDN-1 specific staining is predominantly limited to the basolateral membrane of polarized cells. Scale bars – 20μm.

Figure 2. Prevention and clearance of chronic HCV infection using a CLDN1-specific mAb in vivo

(a, b) Prevention studies. Chimeric uPA-SCID mice received 500 μg CLDN1-specific (n=5) or control mAb (n=4) on days −1, 1 and 5 (arrows) of inoculation (star) with genotype 1b (a) or genotype 4 (b) HCV-containing serum. One infected mouse injected with control mAb died on day 26 (a, cross). (c-e) Treatment studies. Chimeric uPA-SCID mice were chronically infected with HCVcc Jc1 (genotype 2a/2a) (c), HCVcc VL-JFH1 (genotype 1b/2a) (d) or serum of genotype 2a (e). Twenty-four to 50 days following inoculation, the animals received 500 μg control (c, d, e; n=4, 3 and 4 respectively) or CLDN1-specific mAb (c, d, e; n=5, 5 and 6 respectively) each week (arrows) for 4 weeks. Two CLDN1-specific mAb treated mice were sacrificed following viral clearance for FISH studies (e). (f-j) Serum concentration of CLDN1-specific mAb from (a-e respectively) was determined by ELISA. Serum viral load (a-e) was quantified by the clinically licensed Abbott RealTime™ HCV assay. The horizontal dashed line indicates the limit of quantification (LOQ). (c, d, h, i) The viral load and antibody concentration of the mice exhibiting a relapse is indicated by a dashed line. Values of individual animals are shown.

Figure 3. CLDN1-specific mAb impairs HCV-induced host cell signaling

(a) Detection of kinase phosphorylation in chronically HCV Jc1-infected Huh7.5.1 cells treated with control or CLDN1-specific mAbs (100 μg/mL; 24h) using human phosphokinase arrays. (b) p-ERK1/2 highlighted by black squares in (a) was quantified using Image J software (NIH). Results are shown as mean ± s.e.m. of integrated dot blot densities from 2 independent experiments performed in duplicate. (c) ERK1/2 phosphorylation is elevated in liver tissue of patients with chronic HCV infection. Expression of p-ERK1/2, total ERK1/2 and ACTIN was revealed by Western blotting (representative full length blots are presented in Supplementary Fig.13a). (d) Quantification of signal intensities of p-ERK1/2 in (c) normalized to total ERK1/2 expression. The quantification of p-ERK1/2 (normalized to ACTIN expression) relative to total ERK1/2 expression (normalized to ACTIN expression) is shown. Results are shown as the interquartiles (box) with the min and max values (whiskers). The line within the box indicates the median value. (e) Inhibition of the MAPK pathway inhibits persistent HCV infection in Huh7.5.1 cells. Luc-Jc1-infected Huh7.5.1 cells were treated 3 days after inoculation with erlotinib or UO126 (each at 10 μM) for 3 days. HCV replication was assessed by luciferase assay. Results are presented as mean ± s.e.m of 6 independent experiments performed in triplicate. (f) CLDN1-specific mAb reduces ERK1/2 phosphorylation in liver tissue from patients with chronic HCV infection. Fresh liver biopsies were divided in two equal pieces and maintained in DMEM medium supplemented with 10% foetal calf serum and 100 μg/mL control or CLDN1-specific mAb for 4h, respectively. Expression of p-ERK1/2, total ERK1/2 and ACTIN was determined by Western blotting. Information on liver biopsies is provided in Supplementary Table 4 (representative full length blots are presented in Supplementary Fig.13b). (g) CLDN1-specific mAb does not inhibit EGF-induced ERK1/2 phosphorylation. Huh7.5.1 cells were serum starved for 4h prior 1h incubation with 100 μg/mL control or CLDN1-specific mAb and 15 min incubation with increasing concentrations of EGF. P-EGFR, p-ERK1/2 and total ERK1/2 were quantified by Western blotting (full length blots are presented in Supplementary Fig.13c). (h) Quantification of EGF-induced ERK phosphorylation in the presence of CLDN1-specific or control mAb. P-ERK1/2 and total ERK1/2 Western blot signals described in (g) were quantified using a Typhoon Trio laser scanner and ImageQuant Software (GE Healthcare). Values are expressed as relative ratio of p-ERK1/2 to total ERK1/2 densities (two independent experiments ± s.e.m.). (i) Model of HCV-induced signal transduction during cell entry (left panel) and its impairment by CLDN1-specific mAb (right panel). (Left panel) (I) HCV entry is dependent on EGFR signaling which promotes CD81-CLDN1 co-receptor interactions according to Zona et al. and Lupberger et al. (II) At the same time viral binding to the target cell induces EGFR phosphorylation and signaling via CD81-EGFR interactions as shown by Diao et al. and (III) MAPK signaling according to Brazzoli et al. by cross-talk. (Right panel) Combining these observations and results shown in panels (a-h) and Supplementary Fig.10, our findings are consistent with a model that the CLDN1-specific mAb impairs virus-induced MAPK/ERK1/2 signaling by interfering with CD81/CLDN1-MAPK crosstalk without impairing direct EGF-induced ERK1/2 phosphorylation. *p<0.05, **p<0.01, ***p<0.0001 (Student’s t-test (b, e), Mann-Whitney test (d)).

Figure 4. CLDN1-specific mAb leads to elimination of HCV-infected cells from the livers of human chimeric mice in a dose and time-dependant manner

(a) Detection of human hepatocytes and HCV RNA with probes specific for human GAPDH mRNA (green) or HCV Jc1 RNA (red) using FISH. The specificity of the HCV- and GAPDH probes are shown by specific staining of HCV-infected human hepatocytes and absent staining of uninfected human liver (left top panel) and Balb/c mouse liver (right top panel). FISH analyses were then performed in livers of control (left bottom panel) or CLDN1-specific mAb (right bottom panel) treated chimeric uPA-SCID mice. Red arrows indicate HCV positive cells. Scale bar – 50μm. (b) Quantification of the percentage of infected cells in the liver of uninfected chimeric mice (black circles) or HCV Jc1-infected mice treated either four times with control mAb (grey triangles), or once (blue diamonds) or four times (blue circles) with CLDN1-specific mAb. Livers were collected 1 week after the last antibody injection. The quantification was performed after confocal microscopy acquisition. *p<0.05, **p<0.01, ****p<0.0001 (Kruskal-Wallis test).