An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer

Figures

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  Figure 1. Characterization of SARS-CoV-2 antibodies in convalescent patient samples. (A) Serum or plasma antibody reactivity for the 4 SARS-CoV-2 convalescent patients and 1 nonimmune healthy control participant was assessed by ELISA using SARS-CoV-2 S2Pecto, SRBD, SNTD, SARS-CoV S2Pecto, or PBS. Optical density was measured with a 450 nm filter (OD450) using a microplate reader. Error bars indicate the SD; data are representative of at least 2 independent experiments performed in technical duplicate. (B) Plasma or serum neutralizing activity against the VSV-S for SARS-CoV-2 convalescent donor 1989 on days 18, 28, 56, and 90 in a RTCA neutralization assay. Data represent 2 experiments performed in technical duplicate. (C) Plasma or serum neutralizing activity against the WA1/2020 strain of SARS-CoV-2 for convalescent donor 1989 on days 18, 28, 56, and 90 using a FRNT. Data represent experiments performed in technical duplicate.

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  Figure 2. Reactivity, functional, and genetic features of 102 human mAbs identified using LIBRA-Seq. (A) LIBRA-Seq scores for all cells for each experiment are shown as black circles for 3 different LIBRA-Seq runs. Antibodies that demonstrated either full or partial neutralization in the high-throughput RTCA assay are highlighted in green or purple, respectively. (B) mAb specificity or reactivity for each of the 4 S proteins or subdomains. The figure shows a heatmap for binding of 102 mAbs expressed recombinantly, representing OD values collected at 450 nm for each antigen (range, 0.5–4.0). White indicates a lack of detectable binding, blue indicates binding, and darker blue indicates higher OD values. To the right are the antibody numbers that demonstrated either full or partial neutralization in the high-throughput RTCA assay, highlighted in green or purple, respectively. (C) Genetic characteristics of mAbs that demonstrated either full or partial neutralization along with their ELISA reactivity; numbers in the boxes represent OD values collected at 450 nm (range, 0.5–4.0) and LIBRA-Seq scores for each antigen. White boxes indicate no or low reactivity, and red (ELISA) and purple…

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  Figure 3. Activity of neutralizing mAbs against SARS-CoV-2. (A) ELISA binding to SARS-CoV-2 S6Pecto protein was measured by absorbance at 450 nm. Antibody concentrations starting at 20 μg/mL were used and titrated 2-fold. Calculated EC50 values are shown on the graph. Error bars indicate the SD; data represent at least 2 independent experiments performed in technical duplicate. (B) Binding to the surface of VSV-S–infected Vero cells was measured by flow cytometry, and MFI values were determined for dose response binding curves. Antibodies were diluted 3-fold starting from 20 μg/mL. Data represent 2 experiments performed in technical triplicate. (C) VSV-S neutralization curves for mAbs that were expressed after high-throughput RTCA neutralization conformation. Calculated IC50 values are shown on the graph. Error bars indicate the SD; data represent at least 2 independent experiments performed in technical duplicate. (D) Neutralization curves for COV2-3434 or COV2-2196 against SARS-CoV-2 virus. Calculated IC50 values are shown on the graph. Error bars indicate the SD; data represent at least 2 independent experiments performed in technical duplicate. (E) To determine GR COV2-3443 antibody…

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  Figure 4. Epitope identification and structural characterization of COV2-3439 and COV2-3434 antibodies. (A) Competition of the panel of neutralizing mAbs with the previously mapped antibodies COV2-2130, COV2-2196, COV2-2676, COV2-2489, r4A8, and rCR3022. Unlabeled antibodies that were applied to the antigen first are indicated on the left, whereas biotinylated antibodies that were added to antigen-coated wells second are listed across the top. The number in each box represents the percentage of competition binding of the biotinylated antibody in the presence of the indicated competing antibody. Heatmap colors range from dark gray (100% binding of the biotinylated antibody) to white (0% or no binding of the biotinylated antibody). The experiment was performed in biological replicate. A biological replicate from a representative single experiment is shown. (B) nsEM of the SARS-CoV-2 S6Pecto protein in complex with COV2-3439 Fab. Shown are the side view and top view of superimposed 3D volume COV2-3439 Fab–S6Pecto closed trimer (S protein model PDB:7JJI) complexes, as visualized by nsEM for the COV2-2676 Fab model in gold and the COV2-2489 Fab model in gray. At the bottom, negative-stain 2D…

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  Figure 5. Structural characterization of the trimer-disrupting antibody COV2-3434. (A) Residues identified as important for COV2-3434 binding are highlighted as spheres on the S protein structure: Protein Data Bank (PDB) 7L2C (green ribbons), F43 (magenta), F175 and L176 (cyan), and L226 (orange). Residues critical for COV2-3434 binding were identified from binding screens of an alanine-scanning mutagenesis library of NTD. (B) mAb binding values for COV2-3434, COV2-3439, and control anti-NTD mAb COV2-2305 are shown at SARS-CoV-2 S protein clones identified as critical for MAb binding. mAb reactivities for each mutant are expressed as a percentage of binding to wild-type S protein, with ranges (half of the maximum minus minimum values). Two replicate values were obtained for each experiment. (C) nsEM of SARS-CoV-2 rNTD protein in complex with COV2-3439 and COV2-3434 Fabs. Shown are the top view and side view of superimposed 3D volume COV2-3434 Fab–COV2-3439 Fab–SARS-CoV-2 rNTD complexes as visualized by nsEM aligned to the S protein of SARS-CoV-2 in complex with 4A8 (PDB: 7C2L) Data are from a single experiment (detailed collection statistics are provided in Supplemental Table 3). (D) ELISA…

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  Figure 6. Escape virus neutralization and protection in K18 hACE2–transgenic mice by the trimer-disrupting antibody COV2-3434. (A) Neutralization of mAb escape viruses selected by the RBD-specific mAbs COV2-2479 (red), COV2-2130 (green), COV2-2094 (magenta), or COV2-2499 (purple) and the NTD-specific mAbs COV2-2676 (blue) or COV2-2489 (cyan) and with VSV-S by COV2-3434 or COV2-2196 (positive control). The mutations selected by those mAbs are listed with the references. In the right column, the RTCA curves show neutralization of those escape viruses; the asterisk indicates a lack of neutralization in wells with only virus and no antibody. (B) Eight-week-old male K18-hACE2–transgenic mice were inoculated via the i.n. route with 104 FFU of SARS-CoV-2 (WA1/2020 D614G). One day prior to virus inoculation, mice were given a single 200 μg (~10 mg/kg) dose of COV2-3434 or COV2-2196 by i.p. injection. Weight change was monitored daily. Data are from 2 independent experiments (n = 10 per group). **P < 0.01 and ****P < 0.0001, by 2-way ANOVA. Error bars represent the SEM. (C) On day 6 after infection, lungs were collected and assessed for infectious viral burden by plaque assay. PFU/g is shown. Bars…