PMC

Five ml of plasma from each of 5 HCV-infected individuals (Pt1 to Pt5, ) and 3 healthy donors (H1 to H3) was subjected to virus purification for measuring CD59 by Western blot. Pt1 to Pt 5: HCV-infected patients #1 to #5; H1 to H3: healthy donors #1 to #3.

(A) FACS analysis of CD59 surface expression on human hepatocytes (PHHs and Huh7.5.1 cells). (B) PHHs and Huh7.5.1 cells were treated with PI-PLC, and followed by surface and intracellular staining of CD59. (C) Cells were treated (Intra) or untreated (Total) with PI-PLC, and followed by protein extraction for Western blot analysis of CD59 expression. Histograms in red and blue lines show control (isotype) and CD59 staining, respectively. Intra: intracellular CD59; Total: total CD59; Huh7: Huh7.5.1 cells; PHHs: primary human hepatocytes.

(A) ELISA detection of CD59 in cell-free supernatant from: uninfected Huh7.5.1 cells (−HCV); Huh7.5.1 cells infected with 1 MOI of HCV (+HCV) for 5 days; or Huh7.5.1 cells infected with 2 MOI of Ad5 (+Ad5) for 3 days; uninfected THP-1 cells (−HIV-1); THP-1 cells infected with HIV-1 (+HIV-1); and activated U1 cells. (B) Viral particles collected from fractions 1 to 4 were subjected to ELISA, qPCR and Western blot to detect HCV core protein, HCV RNA copies and CD59 expression, respectively. Supernatant from uninfected (−HCV) Huh7.5.1 cells were parallelly tested. (C) Immunocapture of HCV and HIV-1 from cell-free culture supernatant of infected cells by anti-CD59 pAbs. (D) Immunocapture of HCV from the purified HCV virus fraction 3 by anti-CD59 pAbs. Viral RNA copies were quantified by qPCR. All experiments were repeated twice with similar results. @CD59: rabbit anti-human CD59 pAbs.

(A) Virolysis of primary HCV virions. Plasma from HCV-infected patients was directly treated with PBS, IgG (20 µg/ml, BRIC229 (20 µg/ml), rILYd4 (20 µg/ml), or Triton X-100 to trigger autologous ADCML. HCV core released from lysed HCV virions was detected by ELISA. HCV virions treated with Triton X-100 and PBS were used as 100% and blank of virolysis, respectively. (B) The percentage of virolysis was calculated as follows: (core released by CD59 blocker - core released by PBS)/(core released by Triton X-100 - core released by PBS) X 100%. (C) Virolysis data pooled from all HCV plasma samples. Horizontal bars represent mean of virolysis values. Each value represents the mean ± SD of triplicate determinations. The experiments were repeated twice with similar results. **, *, and ≈ indicate p<0.01, p<0.05, and p>0.05, respectively, by the paired Student’s t test.

Cell-free supernatant from HCV (JFH-1)-infected Huh7.5.1 cells was preincubated with IgG, BRIC229 or rILYd4 at 1.25 – 20 µg/ml, followed by addition of anti-HCV E2 pAbs plus complement or heat-inactivated complement. Viral preparations were also treated with PBS and Triton X-100 to determine 0 and 100% lysis, respectively. Virolysis of HCV was quantified by measuring HCV core release using the QuickTiter™ HCV Core ELISA Kit. (A) Dose-dependent analysis of HCV core released from lysed HCV virions in response to BRIC229 or rILYd4 treatment in the presence (solid lines with solid symbols) or absence (dot lines with open symbols) of potent complement plus anti-HCV E2 pAbs. (B) Effects of anti-HCV E2 pAbs on complement-mediated virolysis of HCV virions in response to IgG, BRIC229 or rILYd4 treatment at 20 µg/ml. Complement was added in all conditions with either anti-HCV E2 pAbs (+anti-HCV E2) or anti-HIV-1 gp120/160 pAbs (-anti-HCV E2). (C) Statistical analysis of HCV lysis by ADCML from the experiments described in Fig. 4B. (D) Reduction assay of HCV infectivity. Following the virolysis assays, a reduction assay of viral infectivity was performed by inoculating fresh Huh7.5.1 cells with the conditioned solutions from experiments depicted in the Fig. 4B (+anti-HCV E2). The number of infectious HCV virions remaining in the virolysis solution samples of the ADCML experiments was quantitated by a HCV focus-forming assay. HCV foci were visualized by an immunofluorescence microscopic analysis. Each value represents the mean ± SD of triplicate determinations. ** indicates p<0.01 and ≈ indicates p>0.05, respectively, by paired Student’s t test.