Journal: Advanced Science

Article Title: IgG‐Bridging–Seeded Synergistic Aggregation of SARS‐CoV‐2 Spikes Underlies Potent Neutralization by a Low‐Affinity Antibody

doi: 10.1002/advs.202517192

Figure Lengend Snippet: Neutralization potency, binding affinity, and epitope mapping of antibody P5‐1C8. a‐c) Neutralization c0075rves of P5‐1C8 IgG against SARS‐CoV‐2 WT (a), BA.1 (b), and JN.1 (c) variants. Data were obtained from two independent experiments, each performed with two technical replicates. d–f) Binding kinetics between P5‐1C8 IgG and the spike trimer of SARS‐CoV‐2 WT (d), BA.1 (e), and JN.1 (f), measured by surface plasmon resonance (SPR). Spike trimers were immobilized on a nitrilotriacetic acid (NTA) sensor chip, and serial dilutions of P5‐1C8 IgG were flowed through the system. Colored lines represent experimentally measured sensorgrams. Black lines show the best‐fit curves based on experimental data. The calculated association rate (K a ), dissociation rate (K d ), and equilibrium dissociation constant (K D ) for each antibody‐spike pair are indicated. The dissociation rate constant of P5‐1C8 IgG for WT spike should be interpreted with caution, as it is near the detection limit of the instrument. All results were confirmed in three independent experiments. g) Overall structure of the complex between SARS‐CoV‐2 WT RBD (soft orange) and P5‐1C8 Fab (pink). h) Superimposition of three Fab‐RBD complexes: P2C‐1F11 (dark violet), ZCP3B4 (moderate yellow), and ZCP4C9 (blue). i) Crystal structure of WT RBD (soft orange) in complex with ACE2 (green), panels g‐i show the spatial relationships of all four Fabs relative to the ACE2 binding site. j) Binding footprints of the four Fabs and ACE2 on the SARS‐CoV‐2 RBD. Pink, dark violet, moderate yellow, blue, and green denote the footprints of P5‐1C8 Fab, P2C‐1F11 Fab, ZCP3B4 Fab, ZCP4C9 Fab, and ACE2, respectively. k) Footprint of RBD‐contacting residues on antibody CDR loops, with key amino acids in HCDR1‐3 and LCDR1. CDR positions are annotated according to IMGT numbering. l) Sequence conservation analysis on residues bound by P5‐1C8. These logo plots show the conservation of P5‐1C8 epitopes from SARS‐CoV‐2 WT, Beta, Delta, and Omicron (BA.1, BA.2.75, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1, and JN.1) variants. m) Structure of the SARS‐CoV‐2 WT RBD, with key P5‐1C8 Fab‐binding residues highlighted as violet spheres. n) The interactions between P5‐1C8 Fab and non‐conserved residues in the RBD are affected by strain‐specific mutations during viral evolution. Shown are the structural comparisons of P5‐1C8 Fab interactions with WT RBD (upper panel) and Omicron BA.1 RBD (middle panel), and JN.1 RBD (lower panel) at positions K417N, L455S, F486P, and Y505H. Contacting residues are depicted as sticks, and hydrogen bonds or salt bridges are indicated by dashed lines.

Article Snippet: Codon‐optimized genes encoding trimeric spike proteins of SARS‐CoV‐2 WT, BA.1, BA.5, and JN.1 were synthesized by Tsingke Co., Ltd. and cloned into the SARS‐CoV‐2 S HexaPro vector (Addgene: 154754).

Techniques: Neutralization, Binding Assay, SPR Assay, Sequencing

Journal: Advanced Science

Article Title: IgG‐Bridging–Seeded Synergistic Aggregation of SARS‐CoV‐2 Spikes Underlies Potent Neutralization by a Low‐Affinity Antibody

doi: 10.1002/advs.202517192

Figure Lengend Snippet: Multivalent binding analysis of antibody P5‐1C8 to WT, BA1, and JN1 spike trimers. a) Representative 2D class averages and side views of composite 3D reconstructions from ns‐EM of P5‐1C8 IgG in complex with spike proteins from SARS‐CoV‐2 WT (pink), BA.1(red) and JN.1, at a 3:1 molar ratio (IgG/Spike). In the JN1 S‐P5‐1C8 IgG complex, particles do not form well‐defined complexes but instead aggregate upon binding. Scale bar: 200 nm. b) Representative 2D class averages and side views of composite 3D reconstructions from ns‐EM of P5‐1C8 Fab in complex with spike proteins from SARS‐CoV‐2 WT (blue), BA.1(purple) and JN.1, at a 3:1 molar ratio (Fab/Protomer). No binding is observed in the JN.1 S‐P5‐1C8 Fab complex. c) Semi‐quantitative epitope occupancy analysis derived from ns‐EM shown in (a) and (b), showing the proportion of spike trimers bound by 0, 1, 2–3 Fabs (gray, light cyan, and blue, respectively). Data correspond to the S‐P5‐1C8 IgG (a) and S‐P5‐1C8 Fab (b) complexes for SARS‐CoV‐2 WT, BA.1, and JN.1, as indicated for each participant on the x‐axis. d) Binding affinity values obtained from SPR assays of P5‐1C8 IgG and Fab against SARS‐CoV‐2 WT, BA.1, and JN.1 spikes. The binding of P5‐1C8 Fab to JN.1 spike was not detectable. N.A., not available. All experiments were performed independently in triplicate. e) Cryo‐EM map of the SARS‐CoV‐2 WT spike in complex with P5‐1C8 IgG. The reconstruction reveals two binding stoichiometries: a symmetric complex (1.5 IgG bound) and an asymmetric complex (one IgG bound). Up RBDs, down RBD, and P5‐1C8 Fabs are shown in yellow, grey, and pink, respectively. The Fc region of P5‐1C8, shown in green, was modeled using AlphaFold 3 predictions based on the P5‐1C8 Fc sequence. The absence of Fc density in the WT S‐P5‐1C8 IgG reconstruction is likely due to intrinsic hinge‐mediated flexibility of the IgG molecule. [ ²⁹ ] The zoomed‐in view on the right highlights the structural details within the black dashed box. The two CH1 C‐termini of the Fabs are separated by 38 Å, whereas the N‐termini of the Fc heavy chains are spaced 11 Å apart.

Article Snippet: Codon‐optimized genes encoding trimeric spike proteins of SARS‐CoV‐2 WT, BA.1, BA.5, and JN.1 were synthesized by Tsingke Co., Ltd. and cloned into the SARS‐CoV‐2 S HexaPro vector (Addgene: 154754).

Techniques: Binding Assay, Derivative Assay, Cryo-EM Sample Prep, Sequencing

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: Mass detector parameters used for the analysis of vitamin C, lopinavir, ketoprofen, and valsartan (IS).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques:

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: In vitro hairless mouse skin permeation profiles of lopinavir after the application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) on the mouse skin fixed in the diffusion cells (A) and the arterial plasma concentration versus time profiles of lopinavir after the transdermal application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) in rats (B). Bullet symbols and their error bars represent the means and standard deviations ( n = 3–4). The asterisk (*) represents a value of the RED group significantly different from that of the control group ( p < .05).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: In Vitro, Control, Diffusion-based Assay, Clinical Proteomics, Concentration Assay

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: In vitro skin permeation parameters of lopinavir after the application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) on the hairless mouse skin fixed in the diffusion cells ( n = 3–4).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: In Vitro, Control, Diffusion-based Assay

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: Pharmacokinetic parameters of lopinavir after the transdermal application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) in rats ( n = 3–4).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: Control