Volume 135, Issue 5, Pages 1719-1728.e1 (November 2008) Neutralizing Host Responses in Hepatitis C Virus Infection Target Viral Entry at Postbinding Steps and Membrane Fusion  Anita Haberstroh, Eva K. Schnober, Mirjam B. Zeisel, Patric Carolla, Heidi Barth, Hubert E. Blum, François–Loic Cosset, George Koutsoudakis, Ralf Bartenschlager, Ann Union, Erik Depla, Ania Owsianka, Arvind H. Patel, Catherine Schuster, Françoise Stoll–Keller, Michel Doffoël, Marlène Dreux, Thomas F. Baumert  Gastroenterology  Volume 135, Issue 5, Pages 1719-1728.e1 (November 2008) DOI: 10.1053/j.gastro.2008.07.018 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Neutralizing host responses in HCV infection target HCVpp entry at postbinding steps. (A) Experimental setup. HCVpp binding to Huh7 cells was performed for 1 hour at 4°C in the presence or absence of antibodies. Under these conditions, HCVpp bind to the cells but do not efficiently enter. Synchronous entry and infection occurs when the inoculum is removed and the cells are shifted to 37°C. In protocol I, inhibitory antibodies were added before binding of HCVpp to the target cell. In protocol II, antibodies were added after HCVpp binding to target cells, thus allowing to study the effect of antibodies on viral entry after HCVpp binding. Dashed lines indicate the time intervals at which antibodies were present. Seventy-two hours later, HCVpp infection was assessed by GFP reporter gene expression determination, expressed relative to control infections without addition of inhibitors. (B) Inhibition of HCVpp (1a; H77c strain) entry into Huh7 cells by anti-SR-BI, anti-CD81, antibodies, heparin, or chondroitin sulfate (CSA) in protocols I and II. Mean ± SD of 6 independent experiments is shown. (C) Inhibition of HCVpp (1a; H77c strain) entry into Huh7 cells by purified human anti-HCV IgG (100 μg/mL) from patients with chronic HCV infection or anti-HCV-negative control individuals (CTRL) in protocols I and II. Mean ± SD of at least 4 independent experiments is shown. (D) Dose-dependent inhibition of HCVpp (1a; H77c strain) entry by anti-HCV IgG derived from patient 7 (solid diamonds) or control IgG (open triangles) in experimental protocol II. Mean values ± SD of a representative experiment performed in triplicate are shown. Statistically significant differences (P < .01; t test) in inhibition of HCVpp entry between protocol I and II in panels B and C are indicated (*). Gastroenterology 2008 135, 1719-1728.e1DOI: (10.1053/j.gastro.2008.07.018) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Targeting of HCVpp entry during postbinding steps by monoclonal antienvelope antibodies. (A and B) Inhibition of HCVpp entry (1a; H77c strain) into Huh7 cells by anti-E1 (A) and anti-E2 (B) monoclonal antibodies (mAbs). Inhibition of entry was assessed as described in Figure 1A. (C) Inhibition of HCVpp (1b; HCV J strain) entry by anti-E1 mAbs. (D) Dose-dependent inhibition of HCVpp (1a; H77c strain) entry by anti-E2 mAb AP33 (solid circles), anti-E1 mAb IGH520 (solid squares), or control mouse IgG in experimental protocol II (addition of IgG postbinding). (E) Inhibition of recombinant envelope glycoprotein E1 binding to Huh7 cells by purified anti-E1 antibodies or heparin. Experiments were performed as described in the Materials and Methods section. Cells stained with detection antibodies alone served as negative control (“NC,” light shaded histograms). (F) Inhibition of E1 (subtype 1b), HCV-LP (1a), and HCVpp (1b) binding to Huh7 cells by anti-E1, control IgG, or heparin. Data are shown as percentage of inhibition of binding of ligands incubated with PBS (100%). For panels A–C and F, mean values ± SD of at least 6 experiments are shown. For panels D and E, mean values of a representative experiment performed in triplicate are shown. Statistically significant differences (P < .01; t test) in inhibition of HCVpp entry between protocol I and II (panels A–C) are indicated (*). Gastroenterology 2008 135, 1719-1728.e1DOI: (10.1053/j.gastro.2008.07.018) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Kinetics of anti-HCV IgG-mediated inhibition of HCVpp entry. (A) Schematic drawing of the experimental setup. Inhibition of HCVpp entry into Huh7 cells by purified anti-HCV-IgG derived from patient or control sera (100 μg/mL) was performed as described in legend to Figure 1A, except that patient-derived anti-HCV IgG or control IgG was added every 20 minutes for up to 120 minutes after HCVpp (1a; strain H77c) binding. Dashed lines indicate the time intervals at which antibodies were present. (B) Kinetics of antibody-mediated inhibition of HCVpp (1a; strain H77c) entry by anti-CD81, anti-SR-BI, heparin, and control IgG. (C) Kinetics of antibody-mediated inhibition of HCVpp entry by patient-derived IgG. Purified anti-HCV-IgG derived from patients 1, 7, 8, 9, 12, 17, and 19 (Table 1); IgG derived from an anti-HCV-negative control individual 20 (Table 1), and anti-CD81 mAb were added to HCVpp bound to target cells as depicted in A. Mean values of a representative experiment performed in triplicate are shown. Gastroenterology 2008 135, 1719-1728.e1DOI: (10.1053/j.gastro.2008.07.018) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Inhibition of membrane fusion of HCVpp-lipsomes by patient-derived anti-HCV IgG. The fusion capacities of HCVpp (1a; H77c strain) were tested using lipid mixing assays. The results are expressed as percentages of maximal fluorescence, obtained by addition of Triton X-100 (final 0.1% vol:vol) to the pseudoparticle/liposome suspensions. The experiments were repeated 3 times, and the most representative profiles of the fusion kinetics are presented here. (A) HCVpp were preincubated in the presence or absence of ApoC-I (0.7 μg/mL) or control IgG from control individual 20 (100 μg/mL). (B–D) HCVpp were preincubated in the presence of IgG (100 μg/mL) from anti-HCV-negative control individual 20 or purified anti-HCV IgG (100 μg/mL) from patients 1, 7, 8, 9, 12, 17, and 19. Gastroenterology 2008 135, 1719-1728.e1DOI: (10.1053/j.gastro.2008.07.018) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Inhibition of HCVcc infection by human anti-HCV neutralizing antibodies. (A) Inhibition of Luc-Jc1 HCVcc entry into Huh7.5.1 cells by heparin, anti-CD81, control mouse IgG, anti-SR-BI serum, or control rat serum was studied as described in Figure 1A and the Materials and Methods section. (B) Inhibition of HCVcc entry into Huh7.5.1 cells by human anti-HCV IgG from patients 3, 7, 9, 12, 17, 19, 22, 23, and 24; control IgG from control individual 21 (100 μg/mL); anti-CD81; or anti-SR-BI was performed in protocols I and II as described in Figure 1A. Results are expressed as percentage inhibition relative to control infections performed in the same way but without addition of inhibitors and represent mean ± SD from 4 independent experiments performed in duplicate. Gastroenterology 2008 135, 1719-1728.e1DOI: (10.1053/j.gastro.2008.07.018) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Kinetics of anti-HCV IgG-mediated inhibition of HCVcc entry. (A) Kinetics of inhibition of Luc-Jc1 HCVcc (genotype 2a) entry by anti-CD81 and anti-SR-BI were performed as described in Figure 3A. (B) Kinetics of antibody-mediated inhibition of HCVcc entry by anti-HCV IgG purified from patients with HCV 2a infection. Purified anti-HCV-IgG derived from patients 22, 23, and 24; IgG derived from an anti-HCV-negative control individual 21; and anti-CD81 mAb were added to HCVcc bound to target cells as depicted in Figure 3A. (C) Kinetics of antibody-mediated inhibition of HCVcc entry by patient IgG analyzed previously in the HCVpp system (Figure 4C). Purified anti-HCV-IgG derived from patients 9, 7, 12, and 19 (Figure 3C) were added to HCVcc bound to target cells as described in B. Results are expressed as percentage HCVcc infection relative to infection performed in the absence of antibody and represent mean values from 1 representative experiment performed in duplicate. Gastroenterology 2008 135, 1719-1728.e1DOI: (10.1053/j.gastro.2008.07.018) Copyright © 2008 AGA Institute Terms and Conditions