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hep26430-sm-sup-0001-suppfig1.tif529KFigure S1: Generation of alpaca heavy chain antibodies directed against HCV E2. (A) One alpaca was immunized and boosted with E2e lacking the HVR1 from patient isolate UKN2B2.8 (arrows). Serum samples (ellipses) were collected at day 50 to screen the humoral immune response by ELISA and at day 100 to generate a phage display library expressing nanobodies at the surface. The animal was boosted twice with homologous full-length ectodomain E2e (polygons) . (B) The native conformation of the immunogen was evaluated by ELISA assay using human mAbs recognizing discrete conformation-sensitive epitopes (CBH2, CBH5, CBH7, A8, AR3A, AR3B, AR3C, 1:7, CBH4B, CBH4G and CBH8), and two well-characterized murine mAbs recognizing linear epitopes (AP33 and ALP98). The immunogen was recognized by many of the mAbs that recognize the CD81 binding region (CBH5, A8, AR3A, AR3B, AR3C, 1:7, AP33), as well others that bind to epitopes outside this region (CBH4B, CBH4G, ALP98). (C) Amino acid alignment of the four isolated nanobodies. Cysteines are boxed, the disulfide connectivity is indicated below the alignment, bars show the positions of the CDRs, which are shaded in light grey, according to the IMGT nomenclature . Positions of somatic mutations as revealed by an alignment with the closest homologous germline sequence (IGHV1S1*01) suggested by IMGT V-QUEST and junction analysis are marked by an asterisk below the sequence.
hep26430-sm-sup-0002-suppfig2.tif6123KFigure S2: Functional characterization of nanobodies. (A) E2eΔHVR1 was bound to a StrepTactin Superflow mini column and incubated with nanobody B11 (left panel) or D03 (right panel). Starting material (SM) of the nanobody, wash and elution fractions were analyzed by SDS-PAGE under non-reducing conditions followed by Coomassie Blue staining. (B) Confocal images of biotinylated nanobody reactivity with HEK293T cells transfected to express HCV strain H77 E1E2 glycoproteins. Background fluorescence observed when binding each nanobody to mock transfected cells was subtracted from these images.
hep26430-sm-sup-0003-suppfig3.tif1782KFigure S3: Neutralization of HCV entry by immune sera and recombinant nanobodies. A) Dose-dependent neutralization of HCV H77 pseudoparticles was observed for sera obtained from day 50 following initial immunization (open circles) or day 100 (closed diamonds). No neutralizing activity was observed with a serum sample obtained from an alpaca immunized with the tetanus toxin (open diamonds). B) Neutralization of JFH-1 infectious particles in cell culture. At a dilution of 1/100, the neutralizing potency of serum from day 100 was greater than that of day 50 (Closed diamonds), and no neutralization was observed with the serum from a control animal immunized with tetanus toxin (open triangles). C) Neutralization of genetically diverse pseudoparticles with immune sera. Pre-immune serum (black bars), serum from day 50 (grey bars) and serum from day 100 (white bars) of the immunization schedule were assessed for the breadth of neutralization using patient-isolates in an HCV pseudoparticle entry assay. 1a: UKN1A20.8; 1b: UKN1B12.16; 2a: UKN2A1.2; 2b: UKN2B1.1; 3a: UKN3A13.6; 4: UKN4.11.1; 5: UKN5.15.7; 6: UKN6.5.8. The serum from day 100 consistently neutralized entry to the greatest degree, although some isolates, such as UKN1A20.8, remained resistant to neutralization by the immune sera. (D) Autologous neutralization of recombinant nanobodies was assessed using HCVpp of the immunogen strain UKN2B2.8. Each nanobody was incubated at the indicated concentration with HCVpp expressing UKN2B2.8 E1E2 and used to infect Huh7 cells. Dose-dependent neutralization was observed for D03 (closed squares) and C09, (closed triangles), while B11 (closed circles) and D04 (open triangles) had no effect. D03 was by far the most efficiently neutralizing nanobody, exceeding the neutralization observed for the human anti-E2 mAb 1:7 (closed diamonds) serving as a control. No neutralization was observed with the control anti-tetanus toxin nanobody (open circles).
hep26430-sm-sup-0004-suppfig4.tif1223KFigure S4: Inhibition of E2-CD81 interaction by nanobodies. (A) Linear representation of the epitopes on H77c E2 protein recognized by antibodies used in Fig. 4A. The regions involved in interaction with CD81, as well as the hypervariable regions and transmembrane region are highlighted. Linear epitopes recognized by antibodies used in competition assays are highlighted by sequences of amino acids. The contact residues involved in conformational epitopes are highlighted by colored text: red highlights the overlapping epitopes of mAbs AR3A and 1:7, while orange highlights the epitopes of mAbs AR1A and CBH7. The remaining conformation-sensitive epitopes (CBH4G, AR2A) have not been mapped yet. (B) A panel of well-characterized mAbs with epitopes covering several non-overlapping antigenic regions on HCV E2 were tested in competition assays with nanobody D03. Binding of a half-maximal concentration of each mAb was performed alone (100% binding), or in the presence of D03 at 10µgmL−1. Statistical comparisons were performed using a one-way ANOVA with Bonferroni correction. * denotes p<0.05; ** p<0.01; *** p<0.001. Competition was observed with mAbs 1:7, AR1A, and AR3A, suggesting that the epitope recognized by D03 overlaps the CD81 binding site. (C) Binary complexes (black line) of soluble UKN2b_2.8 E2 ectodomain and nanobodies D03 (upper panel) or B11 (lower panel), respectively, and ternary complexes (light grey line) including CD81-LEL were analyzed by size exclusion chromatography (SEC) in separate runs and the absorption at 280nm is shown. Both ligands were present in molar excess with respect to the HCV E2 ectodomain as indicated by the presence of peak 2. A shift in elution volume of peak 1 suggesting an increase in molecular weight of the complex upon addition of CD81-LEL was observed only for B11 (black arrows), but not for D03. (D) Peaks 1 and 2 from SEC analysis of D03 and B11 samples containing E2 ectodomain, nanobody and CD81-LEL (both ligands in molar excess) were separated by SDS-PAGE followed by Coomassie staining. E2* represents a partially degraded E2 ectodomain. While B11 formed a ternary complex with both E2 and CD81-LEL, no CD81-LEL was found in the D03 complex, demonstrating cross-competition between D03 and CD81-LEL for E2 binding.
hep26430-sm-sup-0005-supptab1.doc46KTable S1. Data collection and refinement statistics for nanobody D03.
hep26430-sm-sup-0006-suppinfo.doc180KSupporting Information

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