Research ArticleHEALTH AND MEDICINE

Proof of concept for rational design of hepatitis C virus E2 core nanoparticle vaccines

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Science Advances  15 Apr 2020:
Vol. 6, no. 16, eaaz6225
DOI: 10.1126/sciadv.aaz6225
  • Fig. 1 Rational design of HCV E2 cores.

    (A) Schematic representation of HCV E2 (amino acids 384 to 746) colored by structural components with VRs in gray, AS412 in pink, FL in cyan, β-sandwich in red, CD81 binding loop in blue, back layer in green, and stalk transmembrane (TM) region in white, and N-linked glycans and conserved disulfide bonds are indicated by green branches and blue dashed lines, respectively. Sequence alignment of the design regions between E2 and E2mc3 is shown below. (B) Structure-based design of E2 mini-cores. Left: Structure of H77 E2c3 (modeled upon H77 E2c in PDB ID: 4MWF) with shortened VR2 loop modeled by LOOPY. The redesigned β-sandwich loop and the shortened VR2 loop are colored in magenta. Disulfide bonds, C494-C564 and C452-C620, which anchor the VR2 loop to the back layer, are shown in yellow sticks. FL, CD81 binding loop, and back layer are also labeled. Middle 1: Structure of H77 E2mc3 with tip-truncated β-sandwich loop and further tVR2 loop is colored in green. Middle 2: RMSF plot for redesigned tVR2 ensemble is shown with the major steps involved in the ensemble-based de novo protein design below. Right: Structure of H77 E2mc3 with five top-ranking tVR2 design variants (E2mc3 v1-v5) colored in pink and highlighted in a transparent molecular surface. (C) SEC profiles of E2mc3 and variants. Left: H77 E2mc3 (in black), v1-v5 (v1 in red and v2-v5 in light red), and v6-v10 (v6 in blue and v7-v10 in light blue). Right: HK6a E2mc3 (in black) and v1 (in red). (D) SDS-PAGE of E2mc3 and variants (left, H77; right, HK6a). (E) EC50 (μg/ml) values of H77 (top) and HK6a (bottom) E2 cores binding to 12 HCV antibodies, including 8 bNAbs (HCV1, HC33.1, HC84.1, AR3C, HEPC3, HEPC74, 212.1.1, and HC1AM), 1 NAb (AR2A), and 3 non-NAbs (AR1A, AR1B, and E1). E2 cores tested here include E2c3, E2mc3, and E2mc3 variants (10 for H77 and 1 for HK6a). (F) Binding affinities [dissociation constant (Kd) values in nM] of H77 and HK6a E2mc3 variants for six selected HCV antibodies. (G) Thermal stability of H77 and HK6a E2c3 and E2mc3 variants measured by DSC. Two thermal parameters, Tm and ΔT1/2, are listed for four H77 E2 cores and three HK6a E2 cores.

  • Fig. 2 Structures of rationally designed HCV E2 cores.

    (A) Crystal structures of H77/HK6a E2mc3 indicate an overall similar fold to H77 E2c and HK6a E2c3 (PDB: 4MWF and 6BKB). (B) Superposition of the β-sandwich loop from the H77 E2mc3-v1 structure on the HK6a E2c3–Fab E1 complex confirms that loss of binding of E2mc3s to Fab E1 results from truncation of the β-sandwich loop. (C) Superposition of E2 of HK6a E2c3 (PDB: 6BKB), H77 E2mc3-v1, H77 E2mc3-v6, and HK6a E2mc3-v1 on the structure of H77 E2c (PDB: 4MWF), illustrating the conformation of the redesigned tVR2 (amino acids 452 to 494). The redesigned tVR2 regions of H77 E2mc3-v1 and HK6a E2mc3-v1 structures are fully modeled but only partly in the H77 E2mc3-v6 structure. (D) Superposition of the H77/HK6a E2mc3 structures to H77 E2c and HK6a E2c3, indicating similar conformation of the neutralization face with only local conformational changes for the redesigned VR2 E2s.

  • Fig. 3 Rational design of self-assembling E2 core nanoparticles.

    (A) Schematic representation of HCV virion (top) and E2 core–based nanoparticle vaccine (bottom). For the HCV virion, single-stranded RNA (SS-RNA), capsid, membrane, and envelope glycoproteins E1 and E2 are labeled, while for the vaccine, the 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 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) nsEM images of SEC-purified H77 E2mc3-v1 nanoparticles. (F) EC50 (μg/ml) values of H77 (top) and HK6a (bottom) E2mc3-v1 nanoparticles binding to 12 HCV antibodies listed in Fig. 1C. (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 to 0.11 μM by twofold dilution for E2mc3-v1 and 52.08 to 1.63 nM by twofold dilution for nanoparticles) and quantitation biosensors, as shown in fig. S5 (H and I). The peak signals (in nanometers) at the highest concentration are listed in the matrix. Higher color intensity indicates greater binding signal measured by Octet.

  • Fig. 4 Immunogenicity of newly designed E2 cores and nanoparticles in mice.

    (A) Schematic representation of the mouse immunization protocol. In study #1, mice were immunized with H77 E2mc3-v1 (group 1), H77 E2mc3-v1-10GS-FR (group 2), and H77 E2mc3-v1-10GS-E2p (group 3). In study #2, mice were immunized with HK6a E2mc3-v1 (group 1), HK6a E2mc3-v1-10GS-E2p (group 2), and HK6a/H77 E2mc3-v1-10GS-E2p mix (group 3). (B) Longitudinal analysis of E2-specific antibody titers in immunized mouse sera at weeks 2, 5, 8, and 11. Top: EC50 titers (fold of dilution) calculated from ELISA binding of mouse sera in study #1 to the coating antigen, H77 E2mc3-v1. Bottom: EC50 titers calculated from ELISA binding of mouse sera in study #2 to the coating antigens HK6a E2mc3-v1 (groups 1–3) and H77 E2mc3-v1 (group 3). Detailed serum ELISA data are shown in fig. S6 (A to D). (C) Mouse serum neutralization in study #1. Top: Percent (%) neutralization of mouse sera against autologous H77 at weeks 2, 5, 8, and 11. Bottom: Percent (%) neutralization of mouse sera against heterologous HCV-1, J6, and SA13 at the last time point, week 11, with an advantage in heterologous NAb responses observed for the E2p group. (D) Mouse serum neutralization in study #2. Percent (%) neutralization of mouse sera against heterologous H77 at weeks 2, 5, 8, and 11. For (B) to (D), the P values were determined by an unpaired, two-tailed Student’s t test in GraphPad Prism 6 and are labeled on the plots, with (*) indicating the level of statistical significance. (E) Validation of the HCVpp neutralization assay using five HCV bNAbs and an HIV-1 bNAb (negative control) against H77. Percent (%) neutralization of all antibodies was determined at three concentrations: 10, 1, and 0.1 μg/ml.

  • Fig. 5 Patterns associated with HCV E2–specific B cell response in mouse immunization.

    (A) Schematic representation of the strategy used to analyze HCV E2–specific B cell response that combines antigen-specific bulk sorting of splenic B cells with NGS and antibodyomics analysis. (B) Statistical analysis of B cell sorting data obtained for group 1 (H77 E2mc3-v1 monomer) and group 3 (H77 E2mc3-v1-10GS-E2p nanoparticle) in study #1. Left: Frequency of E2-specific B cells. Right: Number of E2-specific B cells per million splenic cells. Five mice from group 1 (M1, M3, M5, M6, and M10) and five mice from group 3 (M5, M7, M8, M9, and M10) were randomly selected and analyzed. (C) Distribution of germline gene usage plotted for groups 1 and 3. Top: Germline VH genes. Bottom: Germline Vκ genes. Statistical analysis of number of activated VH/Vκ genes (≥1% of the total population) is shown on the far right. (D) Distribution of germline divergence or degree of SHM plotted for groups 1 and 3. For each group, percent (%) mutation is calculated at the nucleotide level for VH (left) and Vκ (right). Statistical analysis of germline divergence is shown on the far right. (E) Distribution of CDR3 loop length plotted for groups 1 and 3. For each group, CDR3 length calculated at the amino acid (aa) level is shown for heavy (left) and light chains (right). Statistical analysis of RMSF of CDR3 loop length, which is used as an indicator of how much the CDR3 loop length varies within the E2-specific antibodies from each animal. (F) Neutralization curves using purified IgG for groups 1 (left) and 3 (right) in study #1. Autologous H77 (1a) and heterologous SA13 (5a) were tested in HCVpp assays with a starting IgG concentration of 100 μg/ml followed by a series of threefold dilutions. Structural models of the immunogens are placed next to their neutralization curves. (G) Epitope mapping of polyclonal antibody sera from groups 1 and 3 in study #1. Surface model of E2ECTO is shown in the middle with the FL and AS412 colored in cyan and pink, respectively. Statistical analysis of EC50 titers (fold of dilution) of groups 1 and 3 against the FL probe (left) and the AS412 probe (right). Structural models of the designed nanoparticle probes are placed next to their plots. Epitopes on the nanoparticles are colored according to the E2ECTO model. For (B) to (G), statistical analysis was performed using an unpaired, two-tailed Student’s t test in GraphPad Prism 6 and are labeled on the plots, with (*) indicating the level of statistical significance.

Supplementary Materials

  • Supplementary Materials

    Proof of concept for rational design of hepatitis C virus E2 core nanoparticle vaccines

    Linling He, Netanel Tzarum, Xiaohe Lin, Benjamin Shapero, Cindy Sou, Colin J. Mann, Armando Stano, Lei Zhang, Kenna Nagy, Erick Giang, Mansun Law, Ian A. Wilson, Jiang Zhu

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