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1 Supporting Information Giang et al /pnas SI Materials and Methods Cells, Antibodies, and Viruses. Huh-7, Huh-7.5, and Huh (1) as well as 293T cells were grown in DMEM supplemented with 1% (vol/vol) FCS (Invitrogen). Human mabs AR1A, AR1B, AR2A, AR3A, AR3B, AR3C, AR3D (2), CBH-2, CBH-5, CBH- 4B, and CBH-7 (3 5); mouse mabs A4, H53 (6), AP33, AP32, and ALP98 (7 1); and rat mabs 7/59, 9/27, 3/11, 1/39, 2/69A, 7/ 16B, 11/2, and 6/53 (11, 12) have been described elsewhere. The mabs HCV1 (13) and IGH526 (14) were produced by 293 Freestyle cells (Invitrogen) transfected with the pigg1 antibody expression vectors (2) inserted with the corresponding heavy chain variable domain (VH) and light chain variable domain (VL) sequences. The methods for generating hepatitis C virus (HCV) pseudotype virus particles (HCVpp) has been described below, and cell culture-produced HCV (HCVcc) JFH-1 and its chimeras were generated in previous studies (1, 15 17). Exhaustive panning of a HCV-immune phage-display antibody library. The construction of the IgG1κ fragment antigen-binding (Fab) phagemid library was described in a previous study (2). The protocol for human subjects was approved by the Human Subjects Committee for General Clinical Research Center of Scripps Clinic, and informed consent was obtained from the donor. The phagemid library was transformed into Escherichia coli (XL-1 Blue) (Stratagene) by electroporation, and the phage was propagated overnight with VCSM13 helper phage (Stratagene). Exhaustive panning. The panning antigens were presented in various ways according to the exhaustive-panning strategy (Fig. S1). In a panning experiment, antigens were either directly coated or indirectly captured onto a microtiter plate followed by blocking with 4% (wt/vol) nonfat dry milk in PBS. The phage library was added to the wells and incubated for 1 2 h at 37 C, and unbound phages were washed away with PBS/.5% (vol/vol) Tween 2. Bound phages were eluted and used to infect freshly grown E. coli (XL1-Blue) (Stratagene) for titration on LB agar plates with carbenicillin. The phage libraries were panned for four to six consecutive rounds with increasing washing stringency. In the first panning experiment, which was described in our previous study (2), recombinant E2 glycoprotein (genotype 1a, amino acids ) and GST-fused E1E2 masked with Fab fragments B1, C1, or both were used as panning antigens. In the second panning experiment, E1E2 antigens captured by Galanthus nivalis lectin were used, and the results were similar to the first experiment that Fab fragments to AR1 and AR3 were predominantly selected. In the third panning experiment, E1E2 antigens were captured by mab AR3A and masked with mab AR1B, and Fab fragments to AR4 and AR5 were selected. In the fourth and last panning experiment, E1E2 antigens were captured by mab AR3A and masked simultaneously with mabs AR1B, AR4A, and AR5A. E1E2-binding Fab fragments were only isolated after six rounds of selection (instead of the usual three or four rounds), but none of their heavy chains was unique from previous panning experiments. Screening of antigen-specific Fab fragments. To produce soluble Fab fragments, the phage gene III surface protein in fusion with the Fab heavy chain was excised by restriction enzymes SpeI and NheI. The cut phagemids were self-ligated and transformed into XL1-Blue cells where single individual colonies were isolated from the third and fourth round. The colonies were grown in Super Broth medium with carbenicillin overnight at 37 C for the production of soluble Fab fragments by standard protocols (18). The specificities of the Fab fragments were assessed by ELISA, and the DNA sequences of those that bound with high specificity were determined. Conversion of Fab into IgG1. The vector pigg1 encoding the leader sequence and constant region of human IgG1 gene was used for the cloning and expression of full-length IgG1 (2). Vector pigg1 is a derivative of pdr12 (19) in which heavy- and light-chain cloning sites were altered to XhoI/BstEII and SacI/XbaI sites to facilitate direct cloning of the antibody gene fragments. The heavy- and light-chain gene fragments were excised from the phagemids and inserted sequentially into the XhoI/BstEII and SacI/XbaI sites of the vector. The recombinant plasmids were transfected into CHO cells. Stable cell clones were established by selection with L-methionine sulfoxide (MSX) and by limiting dilution. Cell clones expressing high IgG levels were amplified, and the IgGs were purified with a protein A-agarose column (GE Healthcare). ELISA. To study the relationship of different antibodies isolated in this study, a saturating concentration of blocking Fab fragments or IgGs (2 μg/ml) was added to the E1E2 antigens (isolate H77) captured by precoated G. nivalis lectin (5 μg/ml; Sigma) for 3 min before the addition of detecting IgGs or biotinylated IgGs (.5 2 μg/ml). E1E2 antigens were prepared from cell lysates from 293T cells transfected with H77 E1E2 expression plasmid (2). Nonfat milk [4% (wt/vol); Bio-Rad] in PBS and.5% Tween 2 were used as a blocker in assays using lectincaptured antigens. The ELISA plates were washed after a 1-h incubation, and binding of detecting antibodies was detected by HRP-conjugated goat anti-human IgG Fc antibody (1:2,; Pierce) or HRP-conjugated streptavidin (1:2,; Sigma-Aldrich) in PBS with 1% (wt/vol) BSA and TMB substrate (Pierce). The level of inhibition by the blocking antibody was calculated as the percentage reduction of optical density signals produced by the detecting antibody in the presence of the blocking antibody. To study whether the antibodies recognized continuous or discontinuous epitopes, E1E2 antigens were captured onto ELISA wells precoated with lectin (folded protein) or unfolded with.1% (wt/vol) SDS/ mm DTT and incubated at 1 C for 5 min before capture onto ELISA wells (unfolded protein). Binding of the mabs to folded and unfolded proteins was detected by using the peroxidase system. Mouse mab A4 (6), specific for the E1 linear epitope , was used as a positive control. To study the specificity of the antibodies to E1E2 or soluble E2, serially diluted mabs (1-fold dilution from 1 μg/ml) were incubated with E1E2 or E2 antigens without transmembrane region (soluble E2) precaptured by lectin onto microwells. Soluble E2 antigens were produced in cell supernatant from 293T cells transfected with an expression plasmid encoding the soluble E2 cdna or (E ). After a 1-h incubation, the plates were washed, and bound antibodies were detected with HRP-conjugated goat anti-human F(ab ) 2 antibody (1:2,; Pierce) and TMB substrate. To study the apparent affinity of the mabs, serially diluted mabs (twofold dilution from 1 μg/ml) were added to lectincaptured E1E2 antigens for 1 h. The binding of human mabs was detected by HRP-conjugated goat anti-human IgG F(ab ) 2 antibody as above. Noninfected/nontransfected cell lysates were used as negative controls to determine background for each mab. Apparent affinity was defined by the concentration of mabs that produced half of the maximal specific binding in the fitted EC titration curves using GraphPad Prism software. Giang et al. 1of12

2 To study the ability of mab in inhibiting E1E2 binding to CD81, serially diluted mabs (fourfold dilution from 1 μg/ml) were incubated with E1E2 (isolate H77) for 3 min before adding to ELISA wells precoated with the large extracellular loop of CD81 (CD81-LEL) (2 ng per well). After a 1-h incubation, the plates were washed, and binding of E1E2 to CD81-LEL was detected with mabs that do not block CD81 binding (mabs A4, AR2A, and AR1B). mab AR3A is a positive control, and it binds to an epitope overlaps with the CD81 binding site on E2. To map the residues important for forming the antibody epitopes, E1E2 mutant antigens were produced by transfection of 293T cells with the corresponding expression plasmids. The E1E2 antigens in clarified cell lysate were diluted fivefold and captured by lectin as above. The binding signals of the human and control mouse mabs (1 μg/ml) to the alanine mutants were compared with the wild-type H77 E1E2. Mutations that lead to a reduction of >% of binding signals are considered to be critical in the formation of the antibody epitopes. Immunoprecipitation and immunoblot of the E1E2 antigens. Wild-type (H77 isolate) and mutant antigens in transfected 293T cell lysates were incubated with 3 μg of mab IGH526, AR3A, AR4A, AR5A, or an isotype control IgG overnight at 4 C. The control mab IGH526 is specific to E1 (14) and mab AR3A, to E2 (2). Threefold more cell lysate was used for the xn196/35a mutant than for the wild-type E1E2 to compensate for its lower expression level. Protein A-conjugated agarose (2 μl; GE Healthcare) was added to the mixtures and incubated at room temperature for 2 h. The antibody antigen complex was pelleted and washed four times with PBS and.2% (vol/vol) Tween 2. Bound antigens were eluted from the immunoprecipitants by incubating with SDS/PAGE loading buffer supplemented with 2.5 mm DTT at 1 C for 5 min before the samples were analyzed by gel electrophoresis (4 15% gradient SDS/PAGE). The resolved proteins were transferred to an Immobilon-FL membrane (PVDF type; Millipore) by using a semi-dry blotting device (Bio-Rad). The membranes were blocked with the Odyssey Blocking Buffer (LI-COR Biosciences). The immobilized E1 and E2 glycoproteins were detected with the mouse mab A4 (6) and mab HCV1 (13) and the IRDye8CW goat anti-mouse IgG and IRDye7DX anti-human IgG secondary antibodies (diluted 1:1,; LI-COR Biosciences), respectively. The immunoblots were analyzed with the Odyssey Infrared Imaging System and Image Studio software (LI-COR Biosciences). Alanine-scanning mutagenesis. A panel of alanine-scanning mutants of the E2 CD81-binding site was generated previously by Owsianka et al. (21). The other mutants described in this study were generated by using the same E1E2 expression vector as the template (clone H77C) (22). The residues selected for mutagenesis were based on their conservation in an alignment of the E1E2 sequences from 16 HCV isolates from the six major genotypes (H77, H, CON1, OH8, Ch35, U.K.N1b12.6, JFH1, J6, U.K.N2a1.2, U.K. N2b1.1, U.K.N3a1.28, U.K.N , U.K.N5.14.4, U.K.N5.15.7, U.K.N6.5.34, and U.K.N6.5.8). The alanine-scanning mutants were generated with the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) and PCR primers encoding the specific mutations (Integrated DNA Technologies). The mutated sequences were confirmed by DNA sequencing. Glycine was used to substitute conserved alanine residues selected in the study. HCV neutralization assays. The neutralization assays were performed in DMEM supplemented with 1% (vol/vol) FCS (Invitrogen). For HCVcc neutralization, the viruses were propagated by using Huh-7 or Huh-7.5 cells as described previously (1, 23 26). Neutralization of HCVcc-JFH1 and HCVcc-Con1 was conducted at The Scripps Research Institute (TSRI) and the remaining HCVcc at Copenhagen University Hospital, Hvidovre (HVH). The mabs were serially diluted and incubated with HCVcc for 1 h at 37 C, followed by 3-h (HVH) or 6-h (TSRI) infection of cell monolayers. Cells were then washed to remove unbound virus and incubated for an additional 45 h (HVH) or 72 h (TSRI). Infectious foci were stained by anti-ns5a (HVH) or anti-e2 (TSRI) antibodies. For HCVpp neutralization, HCVpp was generated by cotransfection of 293T cells with pnl4-3.lucr E (27, 28) and the corresponding expression plasmids encoding the E1E2 genes at 4:1 ratio by polyethylenimine (29). Virus infectivity was detected with the firefly luciferase assay system (Promega), and percentage neutralization was calculated as residual virus infectivity at the indicated antibody concentrations divided by infectivity without antibody after background subtraction. Background infectivity of the pseudotype virus was determined by using cells transfected with pnl4-3.lucr E only, and pseudotype virus particles displaying the vesicular stomatitis virus envelope glycoprotein G (VSVpp) were a control for nonspecific activity. The E1E2 expression plasmids for the isolates H77, OH8, U.K.N1b5.23, U.K.N1b12.6, J6E3, U.K.N2a1.2, U.K.N2a2.4, U.K.N2b1.1, U.K.N2b2.8, U.K. N3a1.9, U.K.N3a1.28, U.K.N4.11.1, U.K.N , U.K.N5.15.7, and U.K.N have been described previously (9, 2, 3, 31). The KP-S9 isolate (2) was codon-optimized (GenScript) and inserted into the phcmv expression plasmid (32). In a regular neutralization assay, the virus was incubated with the diluted antibodies for 1 h at 37 C before adding to Huh-7 cell monolayers and incubation for another 6 h. To ensure the quality of data for determining virus neutralization, only HCVpp with a signal-to-noise (S/N) ratio (of virus infectivity by HCVpp versus pseudotype virus generated without E1E2) >1 was included. For HCV Envs that produced low levels of HCVpp (KP-S9, U.K. N2b1.1, U.K.N3A1.9, U.K.N5.15.7, and U.K.N6.5.34), the virus antibody mixture was spinoculated onto the cells at 8 g for 1 h. U.K.N was concentrated over a 2% (wt/vol) sucrose cushion before spinoculation. Genetically humanized mouse model (33) for antibody protection studies. Animals and cell lines. Gt(ROSA)26Sor tm1(luc)kaelin (34) (Rosa26- Fluc) mice were obtained from The Jackson Laboratory. Rosa26-Fluc mice contain the firefly luciferase (luc) gene inserted into the Gt(ROSA)26Sor locus. Expression of the luciferase gene is blocked by a loxp-flanked STOP fragment placed between the luc sequence and the Gt(ROSA)26Sor promoter. Cre recombinase-mediated excision of the transcriptional STOP cassette results in luciferase expression in Cre-expressing tissues. Mice were bred and maintained at the Comparative Bioscience Center of The Rockefeller University according to guidelines established by the Institutional Animal Committee. Huh-7.5 and Huh were maintained in DMEM with 1% (vol/vol) FBS and 1% (wt/vol) nonessential amino acids. Production of recombinant adenoviruses. Briefly, adenoviral constructs were transfected into HEK293 cells (American Type Culture Collection) by using the calcium-phosphate method. Transfected cultures were maintained until cells exhibited full cytopathic effect, then harvested and freeze-thawed. Supernatants were serially passaged two more times with harvest at full cytopathic effect and freeze-thaw. For virus purification, cell pellets were resuspended in.1 M sodium phosphate buffer (ph 7.2), lysed in 5% (wt/vol) sodium deoxycholate, and followed by DNase I digestion. Lysates were centrifuged, and the supernatant was layered onto a g/ml CsCl gradient, then spun at 23, rpm on a Beckman Optima 1K-Ultra centrifuge using an SW28 spinning-bucket rotor (Beckman-Coulter). Adenovirus bands were isolated and further purified on a second CsCl gradient with a SW41.Ti spinning-bucket rotor. Resulting purified adenoviral bands were isolated using an 18.5-gauge needle and twice-dialyzed against 4% (wt/vol) sucrose. Adenovirus concentrations were measured at 1 12 times the OD 26 reading on a FLUOstar Omega plate reader (BMG Labtech). Adenovirus stocks were aliquoted and stored at 8 C. HCV generation and infections. Huh or Huh-7.5 cells were electroporated with in vitro-transcribed full-length HCV RNA. Giang et al. 2of12

3 At 72 h postelectroporation, the medium was replaced with DMEM containing 1.5% (vol/vol) FBS, and supernatants were harvested every 6 h starting from 72 h. Pooled supernatants were clarified by centrifugation, filtered through a.45-μm bottle-top filter (Millipore), and concentrated by using a stirred cell (Millipore). Viral titers [% tissue culture infectious dose (TCID )] were determined with Huh-7.5 cells. Bioluminescence imaging. Unless otherwise specified, mice were injected with 1 11 adenoviral particles at 24 h before i.v. injection with TCID HCV-Cre. At 72 h postinfection, mice were anesthetized with ketamine/xylazine and injected i.p. with 1.5 mg of luciferin (Caliper Life Sciences). Bioluminescence was measured with an IVIS Lumina II platform (Caliper Life Sciences). 1. Zhong J, et al. (25) Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 12: Law M, et al. (28) Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. 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(25) Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J Virol 79: Tarr AW, et al. (26) Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33. Hepatology 43: Flint M, et al. (1999) Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol 73: Hsu M, et al. (23) Hepatitis C virus glycoproteins mediate ph-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA 1: Broering TJ, et al. (29) Identification and characterization of broadly neutralizing human monoclonal antibodies directed against the E2 envelope glycoprotein of hepatitis C virus. J Virol 83: Meunier JC, et al. (28) Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus. J Virol 82: Lindenbach BD, et al. (25) Complete replication of hepatitis C virus in cell culture. Science 39: Wakita T, et al. (25) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11: Scheel TK, et al. (211) Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-ns2 by using previously identified mutations. J Virol 85: Barbas CF, III, Burton DR, Scott JK, Silverman GJ (21) Phage Display: A Laboratory Manual (Cold Spring Harbor Lab Press, New York). 19. Burton DR, et al. (1994) Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266: McKeating JA, et al. (24) Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner. J Virol 78: Owsianka AM, et al. (26) Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. J Virol 8: Yanagi M, Purcell RH, Emerson SU, Bukh J (1997) Transcripts from a single full-length cdna clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA 94: Gottwein JM, et al. (27) Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 133: Scheel TK, et al. (28) Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc Natl Acad Sci USA 15: Jensen TB, et al. (28) Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: Failure of homologous neutralizing-antibody treatment to control infection. J Infect Dis 198: Gottwein JM, et al. (29) Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: Role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. 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4 A. Exhaustive panning of an HCV-immune antibody library using E2 and E1E2 antigens Fabs A, B1, B2, B3 & C1. First panning experiment to panning using focus on E2-MAbs panning of pre-selected (results published E2 (aa ). in Law et al. 28 Nature Medicine 14: ) Second panning experiment to focus on E1E2-MAbs 4 rounds of panning using E1E2 antigens captured by Galanthus nivalis lectin. baculovirus-expressed GST-E1E2 (captured by goat anti-gst) masked with Fab B1, C1 or both. 78 of 96 & 94 of 96 clones from Rounds 3 & 4 panning, respectively, bound E1E2 of H77 in ELISA. DNA sequence analysis: (i) Fabs isolated using antigen masked by Fab B1: C1, C1*, C2, C2*, C3, C4, C5, C6, H1, H1*, H1**, H2, H3, I, J1, J2, J3, J3*, J4, L1, L2, L3, L4 & M. (ii) Fabs isolated using antigen masked by Fab C1: B1, D1, D2, D3 & E1. (iii) Fabs isolated using antigen masked by Fab B1 and Fab C1: D1, D1*, D3, D4, E1, F, G & K. DNA sequence analysis: Fabs B3, B3*, B3**, B3***, B4, B5, B6, B7, D1, E1, E2, E3, J5, L5, L6, S2, U2 & U3 (mostly E1). Third panning experiment to focus on non-overlapping E1E2-MAbs 4 rounds of panning using E1E2 antigens captured by MAb AR3A & masked with MAb AR1B. Fourth panning 6 rounds of panning using experiment for more E1E2 antigens captured by non-overlapping MAb AR3A & masked with E1E2-MAbs MAbs AR1B, AR4A & AR5A. 1 of 96 & 19 of 96 clones from Rounds 3 & 4 panning, respectively, bound E1E2 of H77 in ELISA. None of the 192 clones from Rounds 3 & 4 panning, but 4 of 48 clones from Round 6 panning, bound E1E2 of H77 in ELISA. DNA sequence analysis: Fabs N1, N2, N3, N4, O1, P1, P1*, P2, P3, P4, P5, P6, Q1, Q2, R1, S1, T1, U1 & V1. DNA sequence analysis: Fabs B4, E1, E4, L6, L7 & U3. No new panning experiment. The antibody library to E1E2 has been exhausted. B C D Fig. S1. Exhaustive panning of an HCV-immune library with E2 and E1E2 antigens. (A) Experimental scheme and results of the phage-display library panning. (B and C) To isolate rare antibodies from an antibody repertoire (e.g., yellow phage-fab), the antibody library is first panned to isolate dominant antibodies (i.e., red and blue phage-fab). The isolated antibodies are then used to mask their corresponding epitopes on the panning antigens, thus immunofocusing the selection on epitopes that are recognized by subdominant and rare antibodies. The process is repeated until antibodies binding to distinct regions of the antigen are exhausted from the antibody repertoire. (D) The exhaustive-panning strategy allowed the isolation of a panel of mabs recognizing five distinct antigenic regions (ARs) on E1E2 in this study. AR1, AR2, and AR3 are present on monomeric E2 (1), and AR4 and AR5 are found to be present on the E1E2 complex in this study. 1. Law M, et al. (28) Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med 14: 27. Giang et al. 4of12

5 A. Competition using crude Fab BINDING MAb BLOCKING Fab N1 N2 N3 N4 O1 P1 P1a P2 P3 P4 P5 P6 Q1 Q2 R1 S1 T1 U1 V1 AR1BAR2A A AR1A AR1B AR2A AR3A AR3B AR3C AR3D Residual Binding Level -% 26-% 51-% >% B. Crude Fab binding to E1E2 derived from diverse HCV genotypes 2. OD Fig. S2A N1 N2 N3 N4 O1 P1 P1a P2 P3 P4 P5 P6 Q1 Q2 R1 S1 T1 U1 V1 AR3A IgG Fab H77 UKN1B12.16 OH8 CH35 J6E3 JFH UKN2B1.1 UKN3A1.28c UKN UKN UKN6.5.8 Fig. S2. (A) Competition ELISA between crude Fab fragments and purified IgGs to E1E2. Lectin-captured E1E2 antigens were preblocked with undiluted crude Fab fragments for 3 min before the addition of the binding mabs (1 μg/ml for purified mabs and 1:2 for mab A4 hybridoma supernatant). The percentage change in mab binding was calculated as the OD signals in the presence of blocking Fab fragments divided by that of a control crude Fab. (B) Fab binding to E1E2 from diverse HCV genotypes. Undiluted crude Fab fragments were added to lectin-captured E1E2 antigens derived from different genotypes, and bound Fab fragments were detected with peroxidase-conjugated goat anti-human F(ab ) 2 antibody. Results shown are data subtracted with background using a Fab control. EC measurement (Expt 21715) AR1A AR1B AR2A AR3A AR3B AR3C AR3D AR4A AR4B AR5A Goodness of fit (R 2 ) EC (μg/ml) EC (nm) EC measurement (Expt 2111) AR1A AR1B AR2A AR3A AR3B AR3C AR3D AR4A AR4B AR5A Goodness of fit (R 2 ) EC (μg/ml) EC (nm) EC measurement (Expt ) AR1A AR1B AR2A AR3A AR3B AR3C AR3D AR4A AR4B AR5A Goodness of fit (R 2 ) EC (μg/ml) EC (nm) Mean of 3 expts AR1A AR1B AR2A AR3A AR3B AR3C AR3D AR4A AR4B AR5A EC (nm) standard deviation Fig. S3. Apparent affinity (EC ) of anti-e1e2 antibodies. The binding of serially diluted antibodies to lectin-captured E1E2 antigens (isolate H77) was measured in ELISA. The data were fitted into dose response curves for the calculation of the EC values of each antibody by using GraphPad Prism 5 software. The experiments were repeated three times to determine the mean apparent affinity of each antibody. Giang et al. 5of12

6 MAb AR3A MAb AR4A MAb AR5A Tier 1 1 VSVpp S/N = 427,264 1 HCVpp-H77 (genotype 1a) S/N = 26, HCVpp-UKN1b12.6 (genotype 1b) S/N = 34, HCVpp-J6E3 (genotype 2a) S/N = 11, Tier 2 1 HCVpp KP-S9 (genotype 1a) S/N:, 44 1 HCVpp OH8 (genotype 1b) S/N: 49, 17, HCVpp UKN1b5.23 (genotype 1b) S/N: 64, 17, 82 1 HCVpp UKN2A1.2 (genotype 2a) S/N: 35, 11, 57 1 HCVpp UKN2A2.4 (genotype 2a) S/N: 38, 98, HCVpp UKN2b1.1 (genotype 2b) S/N: 22, 36 1 HCVpp UKN2b2.8 (genotype 2b) S/N: 237, 13, 23 1 HCVpp UKN3A1.28 (genotype 3a) S/N: 48, 65, 34 1 HCVpp UKN3A1.9 (genotype 3a) S/N: 53, HCVpp UKN (genotype 4) S/N:,, 12 1 HCVpp UKN (genotype 4) S/N: 119, 8, 42 1 HCVpp UKN (genotype 5) S/N: 21, 2 1 HCVpp-UKN (genotype 6) S/N = 57, Fig. S4. Neutralization of HCVpp by anti-hcv mabs. The antiviral activity of AR4- and AR5-specific antibody was tested against a panel of HCVpp displaying E1E2 from the six major genotypes. VSVpp were used as a control for nonspecific activity. Percentage neutralization was calculated as residual virus infectivity at the indicated antibody concentrations divided by infectivity without antibody. S/N, S/N ratio of virus infectivity by HCVpp versus pseudotype virus generated without E1E2. To ensure the quality of this analysis, only HCVpp with a S/N ratio >1 was included. The HCVpp-U.K.N did not produce a consistent S/N ratio >1 and was excluded. The HCVpp panel in this study is divided into two tiers. The tier 1 HCVpp have good infectivity and can be used diluted and after a freeze-thaw cycle. The tier 2 HCVpp panel has varying infectivity, and KP-S9, U.K.N2b1.1, U.K.N3A1.9, and U.K.N were used fresh with spinoculation, whereas U.K.N was first concentrated over a sucrose cushion before spinoculation. Giang et al. 6of12

7 A 1 HCVcc-JFH1 AR3A AR4A AR5A 1 HCVcc-CON1 %neutralization B %Infection relative to 6 replicates of virus only MAb (µg/ml) 5,5,5,5.5.1 H77/JFH1 J6/JFH1 S52/JFH1 ED43/JFH1 SA13/JFH1 HK6a/JFH1 %Infection relativeto 6 replicates of virus only MAb (µg/ml) 5 1 5,5,5,5 H77/JFH1 J6/JFH1 S52/JFH1 ED43/JFH1 SA13/JFH1 HK6a/JFH1 [AR3A] (µg/ml) [AR4A] (µg/ml) %Infection relative to 6 replicates of virus only ,5,5,5 H77/JFH1 J6/JFH1 S52/JFH1 ED43/JFH1 SA13/JFH1 HK6a/JFH1 % Infection relative to 6 replicates of virus only ,5,5,5 H77/JFH1 J6/JFH1 S52/JFH1 ED43/JFH1 SA13/JFH1 HK6a/JFH1 [AR5A] (µg/ml) [b6] (µg/ml) Fig. S5. Neutralization of HCVcc by anti-hcv mabs. Neutralization of HCVcc-JFH1 and HCVcc-Con1 was conducted at TSRI (A) and the remaining HCVcc at HVH (B). The error bars in B represent the SEM of four replicates; b6 is a negative control antibody. Giang et al. 7of12

8 Average of two neutralization experiments % neutralization total mab (µg/ml) AR3A AR4A AR5A AR3A+AR4A AR3A+AR5A AR4A+AR5A AR3A+AR4A+AR5A 1 1 % neutralization total mab (µg/ml) AR3A AR4A AR3A+AR4A %neutralization total mab (µg/ml) AR3A AR5A AR3A+AR5A 1 1 %neutralization total mab (µg/ml) AR4A AR5A AR4A+AR5A %neutralization total mab (µg/ml) AR3A AR4A AR5A AR3A+AR4A+AR5A Combination Index Median dose Median dose Median dose Median dose Dose Reduction Index (DRI) at IC mab (CI) at IC (AR3A, µg/ml) (AR4A, µg/ml) (AR5A, µg/ml) (total IgG, µg/ml) AR3A AR4A AR5A r AR3A N/A.56 N/A N/A N/A N/A N/A N/A.993 AR4A N/A N/A.74 N/A N/A N/A N/A N/A.987 AR5A N/A N/A N/A 1.1 N/A N/A N/A N/A.993 AR3A/AR4A (1:2) N/A N/A.98 AR3A/AR5A (1:2) N/A N/A AR4A/AR5A (1:1).59 N/A... N/A AR3A/AR4A/AR5A (1:2:2) Fig. S6. Neutralization of HCVpp-H77 by mab combinations. To determine whether mabs AR3A, AR4A, and AR5A could be synergistic in virus neutralization, the mabs were mixed at a constant ratio and titrated against HCVpp-H77. The antibody ratios (1:2 for AR3A to AR4A or AR5A) were selected based on the IC titers of the mabs to this virus. The experiments were performed in triplicate measurement, and the mean results of two experiments were analyzed based on the median-effect equation with CalcuSyn software (1 3). (Top) Overall results. (Middle) Titrations of individual antibody combinations. (Bottom) The results of the analysis are tabulated. Synergy is indicated when the combination index (CI) is less than 1. The dose reduction index (DRI) was calculated by comparing the dose required to reach % virus neutralization when the specified mab was used alone and in combination. The r value is the linear correlation coefficient of the median-effect plot, and a value of 1 indicates perfect conformity of the experimental measurement to the plot. 1. Zwick MB, et al. (21) Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies. J Virol : ter Meulen J, et al. (26) Human monoclonal antibody combination against SARS coronavirus: Synergy and coverage of escape mutants. PLoS Med 3:e Chou TC (26) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58: Giang et al. 8of12

9 Table S1. Antibodies isolated by exhaustive panning of an HCVimmune antibody library Fab VH Gene HCDR3 length (aa) HCDR3 A IGHV ENKFRYCRGGSCYSGAFDM B1, B3, B3*, B3 IGHV DPYVYAGDDVWSLSR B3, B4, B5, B6 & B7 B R---- C1, C1*, C4, C5 & C6 IGHV PETPRYCSGGFCYGEFDN C2 & C2* R C V D1, D1*, D2 & D3 IGHV DPLLFAGGPNWFDH D E1, E2, E3 & E4 IGHV GPYVGLGEGFSE F IGHV GGGTE G IGHV DPGLAINGVVFPYFGLDV H1, H1*, H1 & H2 IGHV SVTPRHCGGGFCYGEFDY H Y I IGHV PHGPGLSLGIYSAEYFDE J1 IGHV VGVRGIILVGGLAMNWLDP J2 --L---VM J3 & J3* --L---T J4 --L---NM F-- J5 --M---T K IGHV DFYIGPTRDVYYGMDV L1, L2, L3 & L4 IGHV AGDLSVGGVLAGGVPHLRHFDP L5, L6 & L7 ----AF---I S---- M IGHV ESLYMIAFGRVIWPPLDY N1, N2 & N4 IGHV5-51 HVPVPISGTFLWREREMHDFGYFDD N L----- O1 IGHV GCLGAKCYYPHYYYGLDV P1 & P1* IGHV GGPAYYTYSDTLTGYHNVVGDY P L---V---F P N-V----AY----F P4, P5, & P L-N-V----Y-----F Q1 & Q2 IGHV DRNSAGGTWLFRDPPPGSTFFDS R1 IGHV DYGVNFGGGSEHNLDY S1 & S2 IGHV DDCRSSTCYLAQHNWQAYYHDS T1 IGHV GGDSSSPYYYPMDV U1 IGHV EVNLKTWNLAHPNVFDV U R-D---I U S------D---I V1 IGHV ENEGEYVWGHFRSDY Symbols denote Fab fragments having the same heavy chains but different light chains. Table S2. Competition ELISA of mabs specific to known epitopes to block binding of mabs AR3A, AR4A, AR4B, and AR5A Detecting IgG (biotinylated) Blocking antibody (mouse, rat or human) m m r r m r r r r r m r m h h h h h AR Domain Domain Domain Domain AR3 A B B C A4 H53 7/59 9/27 AP33 3/11 1/39 2/69A 7/16B 11/2 AP32 6/53 ALP98 CBH-4B CBH-2 CBH-5 CBH-7 AR3A AR3A AR4A AR4B AR5A Residual binding level: %, black; %, dark gray; %, light gray; >%, white. The sources of the blocking mabs are provided in SI Materials and Methods. Giang et al. 9of12

10 Table S3. Mapping of AR4A and AR5A epitopes by alaninescanning mutagenesis mab Mutant Mutation A4 AR4A AR5A 1 xn196/29a xn196/234a xn196/35a xn196/3a xn35/29a xn35/234a xn35/3a Y192A Q193A V194A R195A xn196a xs198a Y21A T24A N25A D26A xn29a xs211a I212A L221A H222A P224A G2A V227A P228A xn234a xs236a W239A P244A V246A T5A L8A R9A H261A D263A V266A A269G S273A A274G Y276A G278A D279A G282A Q289A F291A T292A F293A S294A P295A R296A H298A xn35a xs37a Y39A G311A T314A G315A H316A R317A Giang et al. 1 of 12

11 Table S3. Cont. mab Mutant Mutation A4 AR4A AR5A 61 M318A A319G W32A M322A M323A M324A xn3a W326A xs327a P328A T329A R339A P341A D346A G3A H352A W353A G354A Q412A L413A I414A N415A T416A N417A G418A S419A W42A H421A I422A N423A T4A L427A N428A T435A G436A A439G L441A F442A F447A xn448a R483A P484A Y485A W487A H488A Y489A P491A S1N V6A G523A P5A T526A Y527A W529A G53A N532A D533A T534A D535A V538A N54A Giang et al of 12

12 Table S3. Cont. mab Mutant Mutation A4 AR4A AR5A 122 P545A G547A W549A F5A H617A Y618A P619A T621A xn623a Y624A R63A G634A G635A E637A H638A R639A A642G A643G R657A D658A R659A P664A L665A L666A T669A T67A L6A P676A L682A L685A G688A L689A H691A L692A H693A V697A D698A V699A Q7A Y73A G74A Relative binding level to wild-type E1E2: %, black; %, dark gray; %, light gray; >%, white. The E1E2 mutant panel includes the CD81-binding site mutants developed by Owsianka et al. (1). Additional mutants were generated by site-directed mutagenesis using the same H77c E1E2 expression vector as a template. The panel was expanded to cover selected conserved residues. Mutations that abolish putative N-glycosylation signals are prefixed with x. Mutants 1 7 are double mutants of two E1 glycosylation sites, mutants 8 78 are E1 mutants, and mutants are E2 mutants. The results shown are the mean percentage binding signal of the mabs to the mutants relative to the wild-type E1E2 in at least two independent ELISA experiments. The control mab A4 recognizes the E1 linear epitope Unexpectedly, alanine-scanning mutations of E2 region appear to have an effect on the binding of mab A4 to the E1E2 complex in repeated experiments. Mutations that reduce >% of mab binding (highlighted in black) (at 1 μg/ml) were considered critical residues in the corresponding antibody epitopes. 1. Owsianka AM, et al. (26) Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding. J Virol 8: Giang et al of 12