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. Briefly, Sf9 cells (2 ´ 10⁹) were infected with recombinant baculovirus (bvHCV.Sp7-) at a multiplicity of infection of 5 to 10 and incubated at 27°C for 3 days in Sf900 II serum-free medium (Invitrogen, Carlsbad, CA). Cells were harvested by centrifugation at 2,500 ´ g for 5 min at room temperature. The cell pellet was washed with PBS and resuspended in 18 ml of prewarmed PBS and 3 ml of 90% glycerol containing 10 mM Hepes buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO), and a mixture of EDTA-free protease inhibitors (PI; Roche Applied Science, Indianapolis, IN). The cell suspension was mixed and incubated at 37°C for 5 min. The process was repeated twice so that the final glycerol concentration in the cell suspension reached 30% (1). The cells were then chilled on ice for 5 min and centrifuged at 2,500 ´ g for 10 min at 4°C.

The following purification steps were performed at 4°C. The cell pellet was resuspended with 50 ml of lysis buffer [10 mM Tris·HCl (pH 7.4)/1 mM MgCl₂/1 mM CaCl₂/1 mM PMSF/PI (TNC-PI buffer)] containing 0.5% digitonin and allowed to sit on ice with gentle agitation. When the percentage of cell lysis reached satisfactory levels (as determined by periodic monitoring by trypan blue exclusion), the cell lysate was centrifuged at 26,000 ´ g in an SW28 rotor (Beckman Coulter, Fullerton, CA) for 30 min to remove cell debris. To maximize the HCV-LP yield, the lysis process was repeated by resuspending the cell pellet in fresh lysis buffer containing 0.5% digitonin. The supernatant was loaded onto a 2-ml cushion of 40% OptiPrep (Axis-Shield PoC, Oslo, Norway) in TNC-PI buffer and centrifuged at 160,000 ´ g for 6 h in an SW41 rotor. The pellet was then harvested, suspended in a small volume of 40% OptiPrep in TNC-PI buffer, overlaid with an 8% to 30% gradient of OptiPrep, and centrifuged at 120,000 ´ g for 16 h in an SW41 rotor. One-milliliter fractions were collected. Total protein was quantitated by using Coomassie Plus protein assay reagent (Pierce Biotechnology, Rockford, IL). Fractions were tested for core, E1, and E2 proteins by ELISA and Western blotting, and ultrastructural study of HCV-LP was performed by electron microscopy.

Ninety-six-well Immobilon-P nitrocellulose-bottomed plates (Millipore, Billerica, MA) were coated with monoclonal antibody specific for human IFN-g, IL-2, or IL-4 (BD Biosciences, San Jose, CA) at a concentration of 5 mg/ml in PBS and incubated at 4°C overnight. After five washes with 1´ phosphate-buffered saline/0.05% Tween 20 (PBST), the wells were blocked with 1% BSA (Sigma-Aldrich) for 2 h at 37°C. PBMCs at 2.5 ´ 10⁵ cells per well were added in duplicate and stimulated with soluble protein antigens (2 m g/ml HCV core protein or 4 mg/ml HCV E1/E2 proteins), or HCV peptide pools of core, E1, or E2 (1 mg/ml) in complete RPMI medium 1640 containing 100 units/ml penicillin, 100 mg/ml streptomycin (Mediatech, Herndon, VA), and 5% FCS (Serum Source International, Schuylkill, PA). Negative control was medium with DMSO and positive control was phytohemagglutinin (Sigma-Aldrich) at a concentration of 1 mg/ml. Cells were incubated for 30 h at 37°C, then washed twice with distilled water to lyse the cells. After five washes with PBST, biotinylated anti-human IFN-g or IL-2 detecting antibody (BD Biosciences) was added for 1 h at 37°C. After five washes with PBST, a 1:1,000 dilution of streptavidin peroxidase was added and incubated at room temperature for 1 h followed by five washes with PBST and the addition of 3-amino-9-ethyl carbazole reagent (AEC; Sigma-Aldrich) in substrate buffer. After the spots had developed for 20-25 min, the plates were washed with distilled water to stop the reaction and to wash off the excess substrate, and air-dried. The spots were counted by using a computer-assisted AID ELISPOT Reader System and AID software version 3.5 (Autoimmune Diagnostika, Strassberg, Germany), which is designed to detect spots by predetermined criteria based on size, shape, and colorimetric density. IFN-g, IL-2, or IL-4 SFUs representing single cells were counted and expressed as SFUs per 2.5 ´ 10⁵ PBMCs.

The cells were cultured in duplicate in 96-well flat-bottom plates at 2 ´10⁵ cells per well in 200 ml of RPMI medium 1640 containing 5% heat-inactivated FCS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were stimulated with recombinant HCV core or E1/E2 proteins at a concentration of 2 mg/ml. As negative controls, cells were stimulated with medium alone and as a positive control, phytohemagglutinin was added to the cells at a concentration of 10 mg/ml. Cultures were maintained for 5 days at 37°C in 5% CO₂, then pulsed with [³H]thymidine (GE Healthcare BioSciences, Piscataway, NJ) at 1 mCi per well (1 Ci = 37 GBq). Cells were incubated for an additional 18 h. Subsequently, the cultures were harvested onto MultiScreen fiberglass plates (Millipore). The [³H]thymidine incorporation into DNA was assessed by liquid scintillation counting in an automated b counter (Beckman Coulter). For intrahepatic lymphocyte proliferation, the same protocol was used as for peripheral blood lymphocyte proliferation, but with 3 ´ 10⁵ cells per well. T cell stimulation was expressed as a stimulation index, which was calculated as the average cpm of antigen-driven proliferation per average cpm of medium background. A sample was considered positive when the average stimulation index was greater than 5.

Lymphocytes were isolated from the liver biopsy by mechanical homogenization. Lymphocytes were counted and viability was assessed by trypan blue exclusion. Lymphocytes (3-5 ´ 10⁵) were washed twice with PBS, cultured with 100,000 g-irradiated (3,500 Gy) buffy coat cells, and stimulated with 0.1 mg of anti-human CD3 per ml and 100 units of recombinant human IL-2 per ml in RPMI medium 1640. The lymphocytes were incubated at 37°C and recombinant IL-2 was added every 5 days. After 15 days' incubation, the cells were washed with PBS and stored in liquid nitrogen. The cells were used to test intrahepatic HCV-specific T cell proliferation as described previously.

CD4⁺ and CD8⁺ T cells were purified from PBMCs with magnetic beads (Miltenyi Biotec, Auburn, CA). Irradiated CD4^-CD8^- cells were used as autologous antigen-presenting cells. CD4⁺ or CD8⁺ T cells at a concentration of 1 ´ 10⁵ cells per well were stimulated with HCV core, E1/E2 proteins, and/or HCV OLPs to determine the frequency of IFN-g-producing cells and the lymphoproliferative capacity by ELISPOT and lymphocyte proliferative assay, respectively. Liver-infiltrating lymphocytes from each chimpanzee were expanded from liver biopsies as previously described (2).

1. Sarmiento M, Batterson WW (1992) J Virol Methods 36:151-157.

2. Nascimbeni M, Mizukoshi E, Bosmann M, Major ME, Mihalik K, Rice CM, Feinstone SM, Rehermann B (2003) J Virol 77:4781-4793.