Journal: bioRxiv

Article Title: Rational design of hepatitis C virus E2 core nanoparticle vaccines

doi: 10.1101/717538

Figure Lengend Snippet: (A) Schematic representation of HCV virion (top) and E2 core-based nanoparticle vaccine (bottom). For the HCV virion, single-stranded (SS)-RNA, capsid, membrane, envelope glycoproteins E1 and E2 are labeled, while for the vaccine, optimized E2 core and nanoparticle carrier are labeled. (B) Colored surface models of nanoparticle carriers (top) and E2 core-based nanoparticle vaccines (bottom). Three nanoparticle carriers shown here are 24-meric ferritin (FR) and 60-meric E2p and I3-01. Nanoparticle size is indicated by diameter (in nanometers). (C) SEC profiles of H77 E2mc3-v1 nanoparticles obtained from a Superose 6 10/300 GL column. The particle fraction is indicated by a dotted-line box. While both FR and I3-01 nanoparticles were produced in ExpiCHO cells, E2p nanoparticles were expressed in HEK293 F cells. (D) BN-PAGE of SEC-purified H77 E2mc3-v1 nanoparticles. (E) Negative stain EM images of SEC-purified H77 E2mc3-v1 nanoparticles. ( F ) EC 50 (μg/ml) values of H77 (upper panel) and HK6a (lower panel) E2mc3-v1 nanoparticles binding to 12 HCV antibodies listed in . (G) Antigenic profiles of H77 (left, in red) and HK6a (right, in green) E2mc3-v1 and three nanoparticles against six HCV antibodies. Sensorgrams were obtained from an Octet RED96 using an antigen titration series of six concentrations (3.57-0.11 μM by twofold dilution for E2mc3-v1 and 52.08-1.63 nM by twofold dilution for nanoparticles) and quantitation biosensors, as shown in and . The peak siginals (nm) at the highest concentration are listed in the matrix. Higher color intensity indicates greater binding signal measured by Octet.

Article Snippet: The proteins were further purified by size exclusion chromatography (SEC) on a Superdex 75 Increase 10/300 GL column (GE Healthcare) for E2 cores and on a Superose 6 10/300 GL column (GE Healthcare) for E2p nanoparticles.

Techniques: Labeling, Produced, Purification, Staining, Binding Assay, Titration, Quantitation Assay, Concentration Assay

Journal: bioRxiv

Article Title: Rational design of hepatitis C virus E2 core nanoparticle vaccines

doi: 10.1101/717538

Figure Lengend Snippet: ( A ) SEC profiles of HK6a E2mc3-v1 nanoparticles based on FR, E2p and I3-01 obtained from a Superose 6 200 Increase 10/300 column after immunoaffinity (AR3A) purification. ( B ) BN-PAGE of HK6a E2mc3-v1 nanoparticles based on FR, E2p and I3-01. ( C ) Negative-stain EM images of HK6a E2mc3-v1 nanoparticles based on FR, E2p and I3-01. ( D ) ELISA binding of H77 E2c3-v1 nanoparticles to 12 HCV-specific antibodies. ( E ) EC 50 values (μg/ml) of H77 E2 E2c3-v1 nanoparticles binding to 12 HCV-specific antibodies. ( F ) ELISA binding of HK6a E2mc3-v1 nanoparticles to 12 HCV-specific antibodies. ( G ) EC 50 values (μg/ml) of HK6a E2mc3-v1 nanoparticles with 12 HCV-specific antibodies. In ( E ) and ( G ), EC 50 values were calculated for all ELISA plots in GraphPad Prism 6 except where the highest OD 450 value was below 0.1 or data fitting was ambiguous (denoted as “−”). ( H ) Octet binding of H77 E2mc3-v1 nanoparticles to six HCV-specific antibodies. ( I ) Octet binding of HK6a E2mc3-v1 nanoparticles to six HCV-specific antibodies. In ( H ) and ( I ), sensorgrams were obtained from an Octet RED96 instrument using a titration series of six concentrations (3.57-0.11 μM by twofold dilution for E2mc3-v1 and 52.08-1.63 nM by twofold dilution for E2mc3-v1 nanoparticles) and quantitation biosensors (see Materials and Methods).

Article Snippet: The proteins were further purified by size exclusion chromatography (SEC) on a Superdex 75 Increase 10/300 GL column (GE Healthcare) for E2 cores and on a Superose 6 10/300 GL column (GE Healthcare) for E2p nanoparticles.

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay, Binding Assay, Titration, Quantitation Assay