Fig. 2: In vitro characterization of the HCV-1 sE1E2.Cut1+2.SPYΔN-His6 heterodimer.
a SEC profiles of HEK293F- and ExpiCHO-produced genotype 1a HCV-1, genotype 3 UKNP3.1.2, and genotype 5 UKNP5.2.1 sE1E2.Cut1+2.SPYΔN-His6 antigens. All antigens were purified using a nickel column. Results are shown for three HEK293F and four ExpiCHO production runs. b DSC thermograms of HEK293F- and ExpiCHO-produced HCV-1, UKNP3.1.2, and UKNP5.2.1 sE1E2.Cut1+2.SPYΔN-His6 antigens. All antigens were purified using a nickel column and SEC. Experimental data and Gaussian fits are shown as black dots and red lines, respectively. Thermal denaturation midpoints (Tm or Tm1–3) are labeled. c Site-specific glycan profiles for HCV-1 sE1E2.Cut1+2.SPYΔN-His6 antigens produced in HEK293F and ExpiCHO cells, with two glycan modifications (Kif and Kif/endo F) for the latter. Kifunensine (Kif) was added to ExpiCHO cell cultures to generate oligomannose-type glycans, which were trimmed by endo F1-3 to GlcNAc cores. Glycans are grouped as complex (solid pink), hybrid (pink lines), oligomannose (solid green), or unoccupied (solid gray); undetermined sites are labeled “N.D.” d ELISA-derived EC50 (µg/ml) values for unmodified, Kif-treated, and glycan-trimmed HCV-1 sE1E2.Cut1+2.SPYΔN-His6 binding to 11 NAbs and CD81-Fc. Antigens were produced in ExpiCHO cells and purified by a nickel column and SEC. When OD450 < 0.5 at 10 µg/ml, EC50 values were set to 10 µg/ml. e BLI profiles of unmodified, Kif-treated, and glycan-trimmed HCV-1 sE1E2.Cut1+2.SPYΔN-His6 binding to 10 NAbs (excluding murine NAb A4) and CD81-Fc. Sensorgrams were obtained on an Octet RED96 using a six-point antigen titration series (starting at 1000 nM, followed by 2-fold dilutions) and are shown in Fig. S2g. Peak responses at the highest concentration are summarized in a color-coded matrix (green to red).
