Fig. 4: Impact of design variation on sE1E2 folding, antigenicity, and structure.
a Schematic of five sE1E2 variants derived from the sE1E2.Cut1+2.SPYΔN template. Sequence features used to adjust the anchoring sites (set 1: V1–V3) and C-terminal extensions (set 2: Ext1 and Ext2) are shown on the left and right, respectively. b SEC profiles of three anchoring-site variants (HCV-1 sE1E2.Cut1+2.SPYΔN-V1/2/3-His6; blue, left three) and two extension variants (HCV-1 sE1E2.Ext1/2.Cut2.SPYΔN-His6; red, right two). SEC traces from three production runs of HCV-1 sE1E2.Cut1+2.SPYΔN-His6 (black, dark gray, and light gray) is overlaid for comparison. All antigens were transiently expressed in 25-ml ExpiCHO cultures and purified using a nickel column. c SDS-PAGE analysis of HCV-1 sE1E2 variants. Left: non-reducing gels for anchoring-site variants V2 and V3; right: non-reducing and reducing gels for the extension variant Ext1. SEC fractions 11 and 17–19 were analyzed. Uncharacterized lower-molecular-weight bands are outlined with red boxes. d DSC thermograms of HCV-1 sE1E2 variants, including anchoring-site variants V2 (left) and V3 (middle), and extension variant Ext1 (right). Antigens were purified using a nickel column followed by SEC. Experimental data and Gaussian fits are shown as black dots and red lines, respectively; thermal denaturation midpoints (Tm or Tm1–3) are labeled. e nsEM analysis of HCV-1 sE1E2 variants. Representative 2D class averages and 3D reconstructions of HCV-1 sE1E2.Cut1+2.SPYΔN-His6 (left) and the sE1E2.Cut1+2.SPYΔN-V2-His6 variant (middle), each in complex with AR4A, and HCV-1 sE1E2.Ext1.Cut2.SPYΔN-His6 bound to AR3C and IGH526 (right). Atomic models of sE1E2 and AR4A (PDB ID: 8FSJ) and IGH526 (PDB ID: 4N0Y) were fitted into density maps to aid structural interpretation. The sE1E2.Cut1+2.SPYΔN-His6/AR4A complex is also shown in Fig. 3a. f ELISA-derived EC50 (µg/ml) values for HCV-1 sE1E2 variants binding to 12 NAbs (including IGH526) and CD81-Fc. All sE1E2 variants were produced in ExpiCHO cells and purified using a nickel column and SEC. When OD450 < 0.5 at 10 µg/ml, EC50 values were set to 10 µg/ml.
