Patients. Eighteen patients with ongoing HCV-infection were selected for inoculation experiments in chimeric uPA-SCID mice. Samples were taken early after initial HCV-infection or during persistent infection (ranging from 3 months to 30 years post exposure). HCV-genotyping of the human sera was performed using a Line Probe Assay developed by Innogenetics (Versant® HCV genotype Assay). All patients were vaccinated with HBsAg and non-infected with HIV.
Purification of polyclonal IgG from human plasma samples. Plasma samples, obtained from HCV-infected patients, were heat inactivation (56 °C for 30 minutes) and IgG was purified with a HiTrap Protein G column (GE Healthcare), as described previously (68).
Generation and inoculation of Hu-liv-uPA-SCID mice. Breeding and genotyping of alb/uPA-CBySmn.CB-17 Prkdcscid (uPA-SCID) mice have been described previously (38, 69). SCID mice homozygous for the uPA-transgene were transplanted with cryopreserved human hepatocytes from two HCV-uninfected donors as described previously (38). Only animals with a high repopulation grade, defined by a plasma human albumin (hu-alb) content of 1 mg/mL or greater, were used. Hu-alb levels were quantified with the Human Albumin ELISA Quantitation kit (Bethyl Laboratories Inc.). Human liver-uPA+/+SCID mice were injected intra-peritoneal (IP) or intra-splenic (IS; injection volume technically limited to 100 à 150 µl) with serum/plasma or human B cells, respectively. Animals were sampled weekly and followed until 8 weeks after infection. Mouse plasma samples were stored at -80 °C until analysis. Repopulation characteristics of chimeric mice and quality data of the injected B cell populations can be obtained on request.
Isolation and treatment of human B cells. PBMC were isolated from whole heparinized blood by density gradient centrifugation. Human B cells were positively selected from PBMC using the MACS® Column Technology (Miltenyi Biotec). In brief, anti-CD19 coated microbeads were incubated with PBMC for 15 min at 4 °C. After washing, the cell suspension was loaded onto the prepared MS Column. The unlabeled cells run through while the magnetically labeled cells are retained on the column. After washing, the column is removed from the magnetic field and the magnetically retained cells can be flushed out as the positively selected cell fraction. The purity (≥ 98% required) and viability (≥ 85% required) of the isolated CD19+ cell population was analyzed using flow cytometry. Purified B cells were either left untreated (B) or treated (B_tr) with trypsin and RNase A (70, 71). In brief, B cells were washed with cold PBS and incubated with trypsin (final concentration 0,25%; 37 °C for 15 min). After washing and trypsin-inactivation with soybean trypsin inhibitor (final concentration 0,05%; 37 °C for 10 min), cells were further treated with RNase A (final concentration 0,10%; 37 °C for 15 min) to remove residual viral RNA. After washing, RNase A is inactivated using Protector RNase inhibitor (37 °C for 15 min). Trypsin and trypsin-inhibitor are purchased from Invitrogen; RNase A and protector RNase Inhibitor are purchased from Roche. Method validation is shown in Supplementary Fig. 4.
RNA extraction for in-house RT-PCR amplifications. RNA extraction from serum was done using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics). For total RNA-extraction from non-treated or treated B cells we used the RNeasy Mini Kit (Qiagen). Since B cells could be lost due to the additional wash-steps in the treated preparations, the number of B cells was normalized to the amount of total RNA after extraction (NanoDrop, Thermo Fisher Scientific).
HCV RNA quantification. HCV RNA levels from human serum or mouse plasma were quantified using the COBAS Ampliprep TaqMan HCV Assay (Roche Diagnostics). Mouse plasma samples were tested at a 1/50 dilution, resulting in a limit of quantification (LOQ) of 750 IU/ml. The HCV RNA content of B cells was measured after total RNA extraction from 5 × 105 B cells. cDNA was synthesized using Superscript™ III Reverse Transcriptase (Invitrogen, Life Technologies) with random primers according to standard procedures. Five microliters of cDNA were used for HCV RNA quantification with the COBAS TaqMan HCV Assay, resulting in a LOQ of 46 IU HCV RNA/106 B cells. All extractions and amplifications were run according to universally adopted precautions, such as the use of different rooms for pre-PCR experiments, in order to avoid cross-contamination.
Quasispecies analysis: E1E2 amplification, plasmid generation and sequencing. Quasispecies analysis based on sequencing of the envelope proteins was performed on HCV recovered from human serum, B cells preparations and uPA-SCID plasma. After RNA-extraction, cDNA was synthesized as described and the HCV envelope region was amplified by means of a nested PCR with envelope-specific primers (72). Full-length E1E2 PCR products (containing EcoRV cleavage sites) were purified and cloned in the bacterial pCR®-Blunt vector (Zero Blunt® PCR Cloning Kit, Invitrogen). After plating of transformed cells and plasmid isolation (QIA prep Spin Miniprep Kit, Qiagen), the E1E2-region of the different clones was sequenced using the SANGER technique (ABI3730XL DNA Sequencing System; Applied Biosystems). Amino acid (AA) sequences were deduced and aligned using CLC-DNA workbench (v5.7) and BioEdit Sequence Alignment Editor. Visual inspection of the chromatograms confirmed AA diversity. Maximum-Likelihood phylogenetic trees showing the relationship between E1E2192− 745 sequences were inferred under the LG model (73) of AA substitution with estimated γ-distribution parameter, using PhyML software (74) (v3.0; South of France bioinformatics platform; ATGC). The tree topology (starting with a Neighbor-Joining tree) was optimized using the Nearest Neighbor Interchange (NNI) approach. Bootstrapping with 1000 replicates tested the statistical robustness of the phylogenetic trees. Only branching with a bootstrap value of ≥ 70% was defined as robust clustering.
Generation of expression plasmids coding for patient-derived viral glycoproteins. Expression plasmids coding for E1E2 from viral variants most prevalent in the patient and mouse compartments were generated. After EcoRV-digestion of the bacterial vector, the isolated DNA fragment was cloned into expression vector pcDNA3.1/Hygro (Invitrogen)(ligation using T4 DNA ligase, standard procedures). After transferring the ligation mixture into competent cells, the transformed cells were plated and the correct insert-size and orientation were confirmed by colony PCR. Positive clones were cultivated for plasmid extraction and inserts were sequenced to confirm identity.
EIA to measure human anti-E1E2 antibody responses. To measure E1E2-antibodies recognizing H77 and variant-specific glycoproteins, HCVH77 and variant-specific envelope proteins were generated and used as coating antigens in EIA. To produce glycoproteins, 293-T cells were transfected (CaCl2-method) with expression plasmids and the ensuing cell-lysates were used as source of E1E2-proteins. For EIA, immunoplates were coated overnight with Galanthus nivalis lectin (GNA; Sigma) and blocked with 5% Bovine Serum Albumin (BSA; Sigma). Cell-lysates were allowed to bind for 2 h at room temperature. After washing, human sera were incubated and bound antibodies were visualized with anti-human IgG (Fc)-HRP (goat, Bethyl Laboratories) or anti-human IgM-HRP (goat, Sigma) conjugates and tetramethyl-benzidine (TMB) substrate. Absorbance was measured at 450 nm. For H77-specific IgG detection, sera were serially diluted to determine end-point titers, defined as the highest serum dilution giving a signal above cut-off. The EIA cut-off value (OD ≤ 0,1) was calculated by using serially diluted anti-HCV-negative sera in each assay. A positive control (HCV-positive serum with known anti-E1E2 end-point titer) was included in each assay to test the reproducibility of the assay procedure. The specificity of the EIA was further tested using anti-HCV negative controls, anti-HBsAg positive and anti-EBV positive sera. These sera never showed any reactivity. For IgG-detection to patient-derived variants, sera were assayed at a 1/100 dilution. For IgM-detection to patient-derived variants, sera were assayed at a 1/50 dilution.
Generation of retroviral pseudoparticles (HCVpp), infection and neutralization assay. The production of HCVpp expressing firefly luciferase and their use in neutralization assays were previously described (75–77). Briefly, 293T cells were co-transfected with an envelope-deficient human immunodeficiency virus (HIV) proviral genome expressing luciferase (pNL4.3.Luc.R-E-, NIH) and expression vectors encoding E1E2 glycoproteins (consensus H77c or patient-derived HCV strains), Murine Leukemia Virus (MLV) envelope or an empty vector (‘No-ENV’). The culture supernatant was collected 48 h post transfection, filtered and aliquots were stored at -80 °C. If required, the successful generation of HCVpp was monitored by quantitation of lentiviral associated HIV p24 core protein using a commercially available EIA (QuickTiter™ Lentivirus Titer Kit, Cell Biolabs, San Diego, CA). Prior to neutralisation assay, mixing serial dilutions of HCVpp with Hep3B cells tested the infectivity of the produced HCVpp-stock. At 72 h post infection, cells were lyzed and the luciferase activity of the cells was assayed using the addition of 50 µl Luciferase Assay Substrate (Promega). Relative light units (RLU) were measured in a luminometer (Berthold Centro LB 960). HCVpp stock dilutions resulting in ± 2 × 105 RLU were used as HCVpp dilution in neutralization assays. MLVpp and No-ENVpp served as positive and negative control in infection assays, respectively. For neutralization assays, serial two- to five-fold dilutions of sera were mixed with HCVpp, pre-incubated at 37 °C for 1 hour, and added to Hep3B cells at 37 °C. After 72 hours, luciferase-positive cells were quantified by measuring RLU. Results are reported as IC50 neutralization titer, defined as the sample concentration or dilution conveying 50% reduction in the number of luciferase-positive cells.
Study approvals.
Human study. Participating subjects gave written informed consent and consented to unit blood donation. The Ethical Board of the Ghent University Hospital approved the study protocol (EC # 1994/137).
Animal study. The Animal Ethics Committee of the Faculty of Medicine and Health Sciences of the Ghent University approved the study protocol.