Rescued HCV-associated lymphoma Ids do not react with viral proteins when expressed as soluble IgGs or as cell surface IgMs. (A) Purified J6E2 captured on lectin-coated 96-well plates was incubated with the indicated patients’ Id IgG1s, with anti-E2 mAbs CBH-4G or CBH-5, or with a human IgG1/κ isotype control. Plate-bound IgGs were detected with an HRP-conjugated antihuman IgG. (B) 96-well plates coated with HCV core, NS3, and NS5 antigens were incubated with the indicated patients’ Id IgG1s, with anti-HCV plasma, or with negative control plasma and diluted as indicated. After washing, plate-bound Igs were detected with HRP-conjugated HCV core, NS3, and NS5 antigens. (A-B) Each bar represents the mean optical density of wells incubated with each group of Id IgGs ± the standard deviation. Representative results of 2 experiments for each assay are shown. (C) Single-cell suspensions of 293 T cells transfected with empty pCDM8 vector (filled gray) or with pCDM8 vectors encoding E1E2 of the indicated genotypes were fixed and permeabilized. The cells were then stained with the anti-E2, anti-E1, a human IgG1/κ isotype control mAb, or with a mixture of the indicated patients’ IgG1 containing 0.5 μg of each Id. Cells were then washed, stained with PE-conjugated antihuman IgG, and analyzed by flow cytometry. (D) The patients’ Ids were expressed as human IgMs on the surface of the mouse B-cell line, A20. Positive controls were A20 cells expressing CBH-4B or CBH-4G. Cells were stained with FITC conjugated anti-human IgM (top panel). Cells were incubated for 1 hour on ice with cell culture supernatant containing soluble E2₆₆₁ or mock supernatant. Cells were then washed, stained with AlexaFluor 647-conjugated mouse anti-E2 mAb (H53) (middle panel). Cells were incubated for 1 hour on ice with soluble J6E2 or bovine serum albumin, washed, and further incubated for 1 hour on ice with a 1:1 mixture of the anti-E2 mAbs CBH-2 and CBH-5. After washing, cells were stained with AlexaFluor 647-conjugated antihuman IgG (bottom panel). (C-D) Cells were washed and analyzed by flow cytometry. (E) A20 cells expressing the indicated surface lymphoma patients’ Id IgM or an IgM of irrelevant specificity (SIC5); neutralizing anti-E2 mAbs (CBH2 or CBH5) or a control human IgG1 mAb were incubated with luciferase-reporter HCV produced in cell culture (HCVcc) (J6/JFH(p7-Rluc2A) HCV,¹⁴  titer: 6.3 × 10⁵ TCID₅₀/mL) for 1 hour at 37°C. These samples were then used to inoculate naïve huh-7.5 cells. To measure infectivity, cells were lysed at 48 hours and subjected to standard luciferase assays. Y-axis represents HCVcc infection relative to the A20 SIC5 control. Data represent means and standard deviations (error bars).