Fig. 1: Design and characterization of a recombinant HCV E2E1 nanoparticle immunogen.

a Top: linear representation of full-length E1E2 with the hypervariable region 1 (HVR1), transmembrane domains (TMD), and hydrophobic membrane-proximal external region (MPER) indicated. Bottom: the design of E2E1-I53-50A. The I53-50A.1NT1 (I53-50A) trimerization domain and E1 are separated by a Gly-Ser-rich linker (GSGGSGGSGGSGGS). The amino acid numbers are based on the standard H77 polyprotein numbering¹⁰⁴. The amino acid sequences are shown in Fig. S2 and an overview of the other designs are depicted in Fig. S1a. b NS-EM 2D class averages of SEC-purified E2E1-I53-50A trimers. The E2E1 and I53-50A moieties are indicated. c Site-specific glycan analysis of E2 and E2E1-I53-50A produced in HEK293F cells and purified using Strep-Tactin followed by SEC. The relative occupancy and abundance of the different glycan species per potential N-glycosylation site (PNGS) are indicated in pie diagrams: non-occupied (gray), complex (magenta), hybrid (white/magenta), and oligomannose glycans (green). d Schematic representation of the in vitro assembly of E2E1-I53-50 nanoparticles (E2E1-NP) by mixing E2E1-I53-50A trimers and the pentameric I53-50B subunits. e SEC profiles of E2E1-I53-50A trimers before assembly (blue) and post-assembly (magenta) in a Superose 6 column. E2 monomer is shown for comparison (red). f Reducing and non-reducing SDS gels of E2, E2E1-I53-50A, and E2E1-NP. Representative example of two experiments. g NS-EM images of E2E1-NPs with an example raw NS-EM image left and 2D class averages right. The white scale bar represents 200 nm. h Binding signal data derived from BLI (example curves depicted in Fig. S4a). Monoclonal antibodies (mAbs) were loaded on protein A sensors and binding signal was measured using an equimolar amount of E2 (100 nM). Depicted are the logarithms of the mean area under the curve (AUC) values from two independent experiments. Source data are provided as a Source data file.