Potent neutralizing RBD‐specific antibody cocktail against SARS‐CoV‐2 and its mutant

Abstract The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and its variants has posed a serious global public health emergency. Therapeutic interventions or vaccines are urgently needed to treat and prevent the further dissemination of this contagious virus. This study described the identification of neutralizing receptor‐binding domain (RBD)‐specific antibodies from mice through vaccination with a recombinant SARS‐CoV‐2 RBD. RBD‐targeted monoclonal antibodies (mAbs) with distinct function and epitope recognition were selected to understand SARS‐CoV‐2 neutralization. High‐affinity RBD‐specific antibodies exhibited high potency in neutralizing both live and pseudotype SARS‐CoV‐2 viruses and the SARS‐CoV‐2 pseudovirus particle containing the spike protein S‐RBDV367F mutant (SARS‐CoV‐2(V367F)). These results demonstrated that these antibodies recognize four distinct groups (I–IV) of epitopes on the RBD and that mAbs targeting group I epitope can be used in combination with mAbs recognizing groups II and/or IV epitope to make mAb cocktails against SARS‐CoV‐2 and its mutants. Moreover, structural characterization reveals that groups I, III, and IV epitopes are closely located to an RBD hotspot. The identification of RBD‐specific antibodies and cocktails may provide an effective therapeutic and prophylactic intervention against SARS‐CoV‐2 and its isolates.


｜ In vitro expression of mAbs
The genes encoding the heavy and light chains of the identified antibodies were separately cloned into expression vectors containing IgG1 constant regions, and the vectors were transiently transfected into 293F cells using polyethylenimine (Sigma). After 72 h, the antibodies secreted into the supernatant were collected and captured by protein A Sepharose (GE Healthcare). The bound antibodies were eluted and further purified by gel filtration chromatography using a Superdex 200 high-performance column (GE Healthcare). The purified antibodies were either used in binding or neutralizing assays.

｜ ELISA analysis
ELISA plates were coated with SARS-CoV-2 RBD and S protein at 0.5 μg/mL in phosphate-buffered saline at 4°C overnight. After standard washing and blocking, 100 μL antibody dilution or supernatant of hybridoma clone was added to each well. After 2 h incubation at room temperature, the plates were washed and incubated with 0.08 μg/mL goat anti-human IgG (H + L)/horseradish peroxidase (HRP) for 1 h at room temperature. The reaction was visualized by adding the substrate 3,3  ,5,5  -tetramethylbenzidine (TMB).
Absorbance at 450 nm was measured using an ELISA plate reader.
An ELISA plate was coated with recombinant RBD-His at 1 μg/mL and then treated for 1 h at 37°C with 10 mM DTT followed by washing to determine the effect of disulfide bond reduction on the binding of RBD-specific mAbs. Then, the wells were treated with 50 mM iodoacetamide for 1 h at 37°C. After washing, standard ELISA was performed as described above.
Competitive ELISA was performed to determine the inhibitory activity of RBD-specific mAbs on the binding of biotinylated mAbs to RBD. Briefly, the wells of ELISA plates were coated with the RBD-His tag at 1 μg/mL. A mixture containing 50 μg/mL unlabeled mAb and 1 μg/mL biotinylated mAb was added, followed by incubation at 37°C for 1 h. The binding of biotinylated mAbs was detected after adding HRP-conjugated streptavidin (Zymed Laboratories) and TMB sequentially.

｜ Epitope binding by SPR
For epitope mapping, two different antibodies were sequentially injected and monitored for binding activity using SPR (Biacore T100; GE Healthcare). The sensorgrams showed distinct binding patterns when pairs of testing antibodies were sequentially applied to the purified SARS-CoV-2 RBD or SARS-CoV-2 RBDV367F covalently immobilized onto a CM5 sensor chip. SARS-CoV-2 RBD or SARS-CoV-2 RBDV367F was immobilized to a CM5 sensor chip via amine group for a final RU of approximately 1500. The first antibodies were injected onto the chip until a binding steady state was reached, and the second antibodies were then injected for 180 s. The level of reduction in RU comparing with or without prior antibody incubation is the key criterion to determine that the two mAbs recognize separate or closely situated epitopes.

Viral titers were measured as luciferase activity in relative light units (Bright-Glo Luciferase
Assay Vector System; Promega Biosciences). Neutralization assays were performed by incubating pseudoviruses with serial dilutions of purified mAbs or mAb cocktails at 37°C for 1 h. Cells for SARS-CoV-2 and SARS-CoV-2 pseudovirus (~15,000 per well) were added in duplicate to the virus-antibody mixture. The IC50 of the evaluated mAbs was determined by luciferase activity 48 h after exposure to the virus-antibody mixture using GraphPad Prism 7 (GraphPad Software).

｜ Live SARS-CoV-2 neutralization assay
A neutralization assay of live SARS-CoV-2 was performed using the cytopathic effect (CPE) assay. Briefly, threefold serial dilutions of mAbs were mixed with a volume of 100 PFU authentic SARS-CoV-2 and incubated at 37°C for 1 h. The mixture was added to a monolayer of Vero-E6 cells in a 96-well plate and incubated for 1 h at 37°C. The supernatant was removed, and 200 μL Dulbecco's modified Eagle's medium (supplied with 3% fetal bovine serum) was added to the infected cells. After incubation at 37°C supplied with 5% CO2 for 3 days, all wells were examined for the CPE effect. All experiments were performed in a Biosafety Level 3 facility.

｜ Cryo-EM sample preparation and data collection
Three microliters of the MA1ScFv/MA2Fab/MA5Fab/RBDV367F complex (0.6 mg/mL) were loaded onto a freshly glow discharge (50 s at 20 mA) 1.2/1.3 UltrAuFoil (300 mesh) before plunge freezing using a vitrobot MarKIV (Thermo Fisher Scientific) with a blot force of 0 and 6 s blot time at 100% humidity at 22°C.
Data were acquired on an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter. Movies were collected using SerialEM automated data collection software at a magnification of  22,500 with a pixel size of 1.02 Å. The dose rate was adjusted to 8 counts/pixel/s, and each movie was fractioned in 40 frames of 200 ms. Three thousand micrographs were collected with a defocus range between −0.5 and −3.0 μm.

RBD-specific mAbs that inhibit binding of the RBD to ACE2. (A,B) Serological antibody
responses to the RBD-mFc and RBD-His. The plasma sample from mice six times after vaccination with RBD-mFc was evaluated for binding to the RBD coated on the ELISA plates.
(C,D) Inhibitory curves of each mAb competing with ACE2 binding.

Fig. (S2). Binding activities of RBD-specific mAbs to SARS-CoV-2 RBD by ELISA.
Purified mAbs are applied to ELISA plates coated with trimeric RBD to measure the binding activity.

Fig. (S6). Epitope mapping through competitive binding measured by ELISA and SPR.
His-tagged SARS-CoV-2 RBD was coated onto ELISA plates, and the unlabeled antibody (column) was applied in the presence of each biotinylated antibody (row). The percent reduction of biotinylated antibody binding to RBD was measured as the inhibitory rates shown in this Table. 11 Fig. (S7). mAb epitope mapping through competitive binding measured by SPR. (A) The sensorgrams showed distinct binding patterns when pairs of testing antibodies were 12 sequentially applied to the purified SARS-CoV-2 RBD covalently immobilized onto a CM5 sensor chip. The level of reduction in RU comparing with or without prior mAb incubation is the key criterion to determine two mAbs that recognize separate or closely situated epitopes.
Results are representative of two independent experiments. (B) For epitope mapping, the summary of the mentioned competition in Extended Data Figure 4 is as follows: "+++," >80% competition among same epitopes; "++," 50%-80% overlapping epitopes; "+," 25% to 50% overlapping epitopes; and "−," <25% distinct epitopes. showed distinct binding patterns when pairs of testing antibodies were sequentially applied to the purified RBDV367F, which was covalently immobilized onto a CM5 sensor chip. The level of reduction in RU comparing with or without prior antibody incubation is the key criterion to determine two mAbs that recognize separate or closely situated epitopes. (B) Summary of the abovementioned competition: "+++," >80% competition; "++," 50% to 80%; "+," 25% to 50%; and "−," <25%. Results in (a) are representatives of two independent experiments.     The first screening identified the hybridoma clones binding to RBD with high affinity, and 74 clones were selected. Competitive ELISA was used to determine the clones that reduced the binding of S to the ACE2 receptor by more than 50% (highlighted).