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

Mailly L, Xiao F, Lupberger J, Wilson GK, Aubert P, Duong FH, Calabrese D, Leboeuf C, Fofana I, Thumann C, Bandiera S, Lütgehetmann M, Volz T, Davis C, Harris HJ, Mee CJ, Girardi E, Chane-Woon-Ming B, Ericsson M, Fletcher N, Bartenschlager R, Pessaux P, Vercauteren K, Meuleman P, Villa P, Kaderali L, Pfeffer S, Heim MH, Neunlist M, Zeisel MB, Dandri M, McKeating JA, Robinet E, Baumert TF - Nat. Biotechnol. (2015)

Bottom Line: 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.This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut National de la Santé et de la Recherche Médicale, Unité 1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France. [2] Université de Strasbourg, Strasbourg, France.

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.

No MeSH data available.


Related in: MedlinePlus

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.24 and Lupberger et al.9 (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.23 and (III) MAPK signaling according to Brazzoli et al. by cross-talk22. (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)).
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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.24 and Lupberger et al.9 (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.23 and (III) MAPK signaling according to Brazzoli et al. by cross-talk22. (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)).

Mentions: HCV binding to hepatocellular CD81 and associated receptors activates EGFR and mitogen-activated protein kinase (MAPK) signaling22,23. HCV Jc1 infection increased ERK1/2 phosphorylation and this was reversed by the CLDN1-specific mAb (Fig.3a-b). The significance of HCV-induced MAPK signaling was supported by analyzing liver biopsies from HCV-infected patients (Fig.3c-d, Supplementary Fig.13a, Supplementary Table 4, p< 0.01, Mann-Whitney test). We observed an antiviral effect of small molecule inhibitors targeting MAPK signaling (Fig.3e) at non-toxic concentrations9,24 (Supplementary Table 5). To confirm the effect of the CLDN1-specific mAb on HCV-induced signaling in liver tissue from HCV-infected patients (Supplementary Table 4), freshly isolated liver biopsies were treated ex vivo with CLDN1-specific or control mAb and we observed a reduction of ERK1/2 phosphorylation compared to control-treated biopsies (Fig.3f, Supplementary Fig.13b). These studies show that HCV infection activates MAPK signaling and CLDN1-specific mAb partially reverses this virus-dependant signaling event without impairing ERK activation by the physiological EGFR ligand EGF (Fig.3g-h, Supplementary Fig.13c). These data suggest a model in which the anti-CLDN1 mAb prevents HCV-induced cross-activation of EGFR and ERK by interfering with CD81-CLDN1 association (Fig.3i). Since MAPK signaling has been shown to be relevant for the HCV life cycle, including viral entry24,25, it is likely that this interference contributes to the ability of the antibody to clear an established infection.


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

Mailly L, Xiao F, Lupberger J, Wilson GK, Aubert P, Duong FH, Calabrese D, Leboeuf C, Fofana I, Thumann C, Bandiera S, Lütgehetmann M, Volz T, Davis C, Harris HJ, Mee CJ, Girardi E, Chane-Woon-Ming B, Ericsson M, Fletcher N, Bartenschlager R, Pessaux P, Vercauteren K, Meuleman P, Villa P, Kaderali L, Pfeffer S, Heim MH, Neunlist M, Zeisel MB, Dandri M, McKeating JA, Robinet E, Baumert TF - Nat. Biotechnol. (2015)

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.24 and Lupberger et al.9 (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.23 and (III) MAPK signaling according to Brazzoli et al. by cross-talk22. (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)).
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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.24 and Lupberger et al.9 (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.23 and (III) MAPK signaling according to Brazzoli et al. by cross-talk22. (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)).
Mentions: HCV binding to hepatocellular CD81 and associated receptors activates EGFR and mitogen-activated protein kinase (MAPK) signaling22,23. HCV Jc1 infection increased ERK1/2 phosphorylation and this was reversed by the CLDN1-specific mAb (Fig.3a-b). The significance of HCV-induced MAPK signaling was supported by analyzing liver biopsies from HCV-infected patients (Fig.3c-d, Supplementary Fig.13a, Supplementary Table 4, p< 0.01, Mann-Whitney test). We observed an antiviral effect of small molecule inhibitors targeting MAPK signaling (Fig.3e) at non-toxic concentrations9,24 (Supplementary Table 5). To confirm the effect of the CLDN1-specific mAb on HCV-induced signaling in liver tissue from HCV-infected patients (Supplementary Table 4), freshly isolated liver biopsies were treated ex vivo with CLDN1-specific or control mAb and we observed a reduction of ERK1/2 phosphorylation compared to control-treated biopsies (Fig.3f, Supplementary Fig.13b). These studies show that HCV infection activates MAPK signaling and CLDN1-specific mAb partially reverses this virus-dependant signaling event without impairing ERK activation by the physiological EGFR ligand EGF (Fig.3g-h, Supplementary Fig.13c). These data suggest a model in which the anti-CLDN1 mAb prevents HCV-induced cross-activation of EGFR and ERK by interfering with CD81-CLDN1 association (Fig.3i). Since MAPK signaling has been shown to be relevant for the HCV life cycle, including viral entry24,25, it is likely that this interference contributes to the ability of the antibody to clear an established infection.

Bottom Line: 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.This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut National de la Santé et de la Recherche Médicale, Unité 1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France. [2] Université de Strasbourg, Strasbourg, France.

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.

No MeSH data available.


Related in: MedlinePlus