A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses.

The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2-36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2-36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378 T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2-36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.

Introduction

Coronaviruses are zoonotic pathogens found in avian and mammalian reservoirs, and seven strains have been found to spillover to humans. Among them, four continually circulate in the human population and only cause mild symptoms of the common cold: 229E and NL63 belong to the alpha-coronavirus genus and OC43 and HKU1 belong to the beta-coronavirus genus [1]. The other three human coronaviruses are all highly pathogenic and belong to the beta-coronavirus genus: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the current COVID-19 pandemic, and SARS-CoV, which caused an outbreak 18 years ago, are members of the subgenus sarbecovirus; whereas Middle-East respiratory syndrome coronavirus (MERS-CoV) is a member of the merbecovirus subgenus [2].

Phylogenetic analysis of the entire genomes grouped SARS-CoV-2 and SARS-CoV with some SARS-related coronaviruses found in bats or pangolins, including bat coronaviruses RaTG13, Rs4231, SHC014, and WIV1, as well as pangolin coronaviruses Pangolin Guangdong and Pangolin Guangxi in the Sarbecovirus subgenus [2]. Both SARS-CoV-2 and SARS-CoV express a transmembrane glycoprotein termed spike protein, which mediates viral entry into host cells by engaging ACE2 as the receptor [3,4] and is, therefore, the primary target of virus-neutralizing antibodies. There is also experimental evidence showing that some of these bat or pangolin viruses could enter into human cells expressing ACE2 [5], indicating their pandemic potential.

SARS-CoV-2 is the causative agent of COVID-19, having infected >238 million people and caused >4.8 million deaths worldwide. Over the past year, several protective vaccines and neutralizing antibody-based therapeutics have become available. However, the emergence of SARS-CoV-2 variants has altered the landscape, threatening the efficacy of these interventions. We and others have shown that some variants such as B.1.351 [6], P.1 [7], B.1.526 [8] and B.1.427/B.1.429 [9] are more resistant to neutralization by some mAbs, as well as by sera from convalescent patients and vaccines. As an example, a single mutation, E484 K, found in several variants could knock out a class of antibodies binding the receptor binding motif (RBM) on the viral spike [6-8]. Therefore, finding a reagent that can target not only the SARS-CoV-2 mutant variants but also related sarbecoviruses is of utmost importance.

Here we describe the isolation of a mAb that cross-reacts and broadly neutralizes SARS-CoV-2 variants, SARS-CoV, and a panel of bat and pangolin sarbecoviruses. Structural analyses and in vitro escape mutation selection indicate that this mAb targeting a highly conserved RBD epitope that could be informative for the development of pan-sarbecovirus vaccines and therapeutics.

Materials and methods

Cell lines

HEK293T/17 (cat# CRL-11268) and Vero E6 cells (cat# CRL-1586) were from ATCC, 293T-ACE2 cells were kindly provided by J. Sodroski of Harvard Medical School, and they were cultured in 10% fetal bovine serum (FBS, GIBCO cat# 16140071) supplemented Dulbecco’s Modified Eagle Medium (DMEM, ATCC cat# 30-2002) at 37°C, 5% CO2. I1 mouse hybridoma cells (ATCC, cat# CRL-2700) were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC cat# 30-2003) with 20% FBS.

Pseudovirus neutralization assays

Plasmids encoding the single-mutation and the combination of mutations found in SARS-CoV-2 variants were generated by Quikchange II XL site-directed mutagenesis kit (Agilent). Recombinant Indiana vesicular stomatitis virus (VSV) expressing different coronavirus spikes were generated as previously described [10,11]. Briefly, HEK293 T cells were grown to 80% confluency before transfection with the spike gene using Lipofectamine 3000 (Invitrogen). Cells were cultured overnight at 37°C with 5% CO2, and VSV-G pseudo-typed ΔG-luciferase (G*ΔG-luciferase, Kerafast) was used to infect the cells in DMEM at a multiplicity of infection (MOI) of 3 for 2 hrs before washing the cells with 1X DPBS three times. The next day, the transfection supernatant was harvested and clarified by centrifugation at 300 g for 10 min. Each viral stock was then incubated with 20% I1 hybridoma (anti-VSV-G, ATCC: CRL-2700) supernatant for 1 hr at 37°C to neutralize contaminating VSV-G pseudo-typed ΔG-luciferase virus before measuring titers and making aliquots to be stored at −80°C. Neutralization assays were performed by incubating each pseudovirus with serial dilutions of a mAb and scored by the reduction in luciferase gene expression as previously described [10,11]. Briefly, Vero E6 cells (for SARS-CoV-2 and SARS-CoV) or 293T-ACE2 cells (for bat/pangolin coronaviruses) were seeded in 96-well plates (2 ×104 cells per well). Each pseudovirus was incubated with serial dilutions of a mAb in triplicate for 30 min at 37 °C. The mixture was added to cultured cells and incubated for an additional 16 hrs. Luminescence was measured using Luciferase Assay System (Promega), and IC50 was defined as the dilution at which the relative light units were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background in the control groups with cells only. The IC50 values were calculated using a five-parameter dose–response curve in GraphPad Prism v.8.4.

Authentic SARS-CoV-2 microplate neutralization

The SARS-CoV-2 viruses USA-WA1/2020 (WA1), hC0V-19/USA/CACDC_5574/2020 (B.1.1.7), hCoV-19/South Africa/KRISP-K005325/2020 (B.1.351), hCoV-19/Japan/TY7-503/2021 (P.1), and hCoV-19/USA/NY-NP-DOH1/2021 (B.1.526) were obtained from BEI Resources (NIAID, NIH). The viruses were propagated for one passage using Vero E6 cells. Virus infectious titer was determined by an end-point dilution and cytopathic effect (CPE) assay on Vero E6 cells as described previously [10,11]. An end-point-dilution microplate neutralization assay was performed to measure the neutralization activity of purified mAbs. Triplicates of each dilution were incubated with SARS-CoV-2 at an MOI of 0.1 in EMEM with 7.5% inactivated FBS for 1 hr at 37°C. Post incubation, the virus-antibody mixture was transferred onto a monolayer of Vero E6 cells grown overnight. The cells were incubated with the mixture for ∼70 h. CPE was visually scored for each well in a blinded fashion by two independent observers. The results were then converted into percentage neutralization at a given sample dilution or mAb concentration, and the averages ± SEM were plotted using a five-parameter dose–response curve in GraphPad Prism v.8.4.

SARS-CoV neutralization assay

Antibodies were subjected to successive two-fold dilutions starting from 20 μg/ml. Quadruplicates of each dilution were incubated with SARS-CoV GZ50 strain (GenBank accession no. AY304495) at MOI of 0.01 in DMEM with 2% inactivated FBS for 1 h at 37°C [12]. After incubation, the virus-antibody mixture was transferred onto a monolayer of Vero E6 cells grown overnight. The cells were incubated with the mixture for 72 h. Cytopathogenic effects of viral infection were visually scored for each well in a blinded manner by two independent observers. The results were then converted into the percentage of neutralization at a given monoclonal antibody concentration, and the data were plotted using a five-parameter dose–response curve in GraphPad Prism v.8.4.

Protein expression and purification

The SARS-CoV-2 and SARS-CoV S2P spike constructs were produced as previously described [3]. The proteins were expressed in HEK293 Freestyle cells (Invitrogen) in suspension culture using serum-free media (Invitrogen) and transfected into HEK293 cells using polyethyleneimine (Polysciences). Cell growths were harvested four days after transfection, and the secreted proteins were purified from supernatant by nickel affinity chromatography using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare) in 10 mM Tris, 150 mM NaCl, pH 7.4. Spike purity was assessed by SDS-PAGE. 2–36 was expressed and purified as previously described [10]. Fab fragments were produced by digestion of IgGs with immobilized papain at 37°C for 3 h in 50 mM phosphate buffer, 120 mM NaCl, 30 mM cysteine, 1 mM EDTA, pH 7. The resulting Fabs were purified by affinity chromatography on protein A, and purity was assessed by SDS-PAGE.

ELISA

ELISA detection of mAbs binding to SARS-CoV-2 and SARS-CoV spike trimers was performed as previously described [10]. For the competition ELISA, purified mAbs were biotin-labelled using One-Step Antibody Biotinylation Kit (Miltenyi Biotec) following the manufacturer’s recommendations and purified using 40 K MWCO Desalting Column (ThermoFisher Scientific). Serially diluted competitor antibodies (50 μl) were added into spike trimer-precoated ELISA plates, followed by 50 μl of biotinylated antibodies at a concentration that achieves an OD450 reading of 1.5 in the absence of competitor antibodies. Plates were incubated at 37 °C for 1 h, and 100 μl of 500-fold diluted Avidin-HRP (ThermoFisher Scientific) was added into each well and incubated for another 1 h at 37 °C. The plates were washed with PBST between each of the previous steps. The plates were developed afterwards with 3,3’,5,5'-tetramethylbenzidine (TMB) and absorbance was read at 450 nm after the reaction was stopped. For the ACE2 competition ELISA, 100 ng of ACE2 protein (Abcam) was immobilized on the plates at 4 °C overnight. The unbound ACE2 was washed away by PBST and then the plates were blocked. After washing, 100 ng of S trimer in 50 μl dilution buffer was added into each well, followed by addition of another 50 μl of serially diluted competitor antibodies and then incubation at 37 °C for 1 h. The ELISA plates were washed four times with PBST and then 100 μl of 2,000-fold diluted anti-strep-HRP (Millipore Sigma) was added into each well for another 1 hr at 37 °C. The plates were then washed and developed with TMB, and absorbance was read at 450 nm after the reaction was stopped.

Surface plasmon resonance (SPR)

The antibody binding affinity to SARS-CoV-2 and SARS-CoV spike trimers and RBDs was detected by Biacore T200 SPR system (Cytiva). All experiments were performed at 25°C in HBS-EP+ buffer (10 mM HEPES, pH 7.4; 150 mM NaCl; 3.4 mM EDTA; 0.005% (v/v) surfactant P20). The anti-His tag antibodies, diluted at 50 µg/mL in 10 mM sodium acetate, pH 4.5, were immobilized on both the active and reference flow cells surface of the activated CM5 sensor chip using amine coupling method. Approximately 200 RU of His-tagged SARS-CoV-2 and SARS-CoV spike trimers and RBDs were captured onto the chip for the active surface, and anti-His antibody alone served as the reference surface. The antibodies were injected through both flow cells at different concentrations (ranging from 300 to 1.2 nM in 1:3 successive dilutions) at a flow rate of 30 µL/min for 120 s, followed by a 15 s dissociation step. After each assay cycle, the sensor surface was regenerated with a 30 s injection of 10 mM glycine, pH 1.5, at a flow rate of 30 µL/min. Background binding to reference flow cells was subtracted and antibody binding levels were calculated using Biacore T200 evaluation software (GE Healthcare).

Cryo-EM grid preparation

Samples for cryo-EM grid preparation were produced by mixing purified spike protein to a final trimer concentration of 0.33 mg/mL with 2–36 Fab in a 1:9 molar ratio, followed by incubation on ice for 1 h. The final buffer for the 2–36 complex was 10 mM sodium acetate, 150 mM NaCl, pH 5.5. n-Dodecyl β-D-maltoside (DDM) at a final concentration of 0.005% (w/v) was added to the mixtures to prevent preferred orientation and aggregation during vitrification. Cryo-EM grids were prepared by applying 3 µL of sample to a freshly glow-discharged carbon-coated copper grid (CF 1.2/1.3 300 mesh); the sample was vitrified in liquid ethane using a Vitrobot Mark IV with a wait time of 30 s, a blot time of 3 s, and a blot force of 0.

Cryo-EM data collection and analysis

Cryo-EM data for single particle analysis was collected on a Titan Krios electron microscope operating at 300 kV, equipped with an energy filter and a Gatan K3-BioQuantum direct detection detector, using the Leginon [13] software package. Exposures were taken with a total electron fluence of 41.92 e-/Å2 fractionated over 60 frames, with a total exposure time of 3 s. A defocus range of −0.8 to −2.5 µm was used with a magnification of 81,000x, and a pixel size of 1.07 Å.

Data processing was performed using cryoSPARC v2.15 [14]. Raw movies were aligned and dose-weighted using patch motion correction, and the CTF was estimated using patch CTF estimation. Micrographs were picked using blob picker, and a particle set was selected using 2D and 3D classification. The resulting particle set was refined to high resolution using a combination of heterogenous and homogenous refinement, followed by nonuniform refinement. The interface between RBD and 2–36 Fab was locally refined by using a mask that included RBD and the variable domains of the Fab. The final global and local maps were deposited to the EMDB with ID: EMD-24190.

Model building and refinement

The 2-36-RBD complex model was built starting from template PDB structures 6BE2 (Fab) and 7BZ5 (RBD) using Phenix Sculptor. SARS-CoV-2 S2P spike density was modelled starting with PDB entry 6VXX [15]. Automated and manual model building were iteratively performed using real space refinement in Phenix [16] and Coot [17]. 2–36 Fab residues were numbered according to the Kabat numbering scheme. Geometry validation and structure quality assessment were performed using Molprobity [18]. PDBePISA was used to calculate buried surface area [19]. A summary of the cryo-EM data collection, processing, and model refinement statistics is shown in Table S1. The final model was deposited in the PDB with ID 7N5H.

Structure conservation analysis

The conservation of each RBD residue was calculated using the entropy function of the R package bio3d (H.norm column). The calculation was based on the sequence alignment of SARS-CoV, SARS-CoV-2 and SARS-related bat coronavirus. The visualization of sequence entropy was displayed by PyMol version 2.3.2.

In vitro selection for resistant mutations against mAb 2–36

SARS-Cov-2 isolate USA-WA1/2020 was mixed with serial five-fold dilutions of 2–36 antibody at MOI 0.2 and incubated for 1 h. Following incubation, the mix was overlaid on 24-well plate to a final volume of 1 mL. the plates were incubated at 37°C for 70 h till CPE was complete (100%) in virus control wells bearing no antibody. At this time, all wells were scored to determine the 50% inhibition titer (EC50) and supernatant collected from this well was used for subsequent round of selection. Passaging continued till the virus was able to form CPE in the presence of 50 µg/mL of 2–36 antibody. At this point, the resulting supernatant was collected, and RNA was extracted using QiaAMP Viral RNA kit (Qiagen). cDNA was obtained using Superscript IV enzyme (Thermo Scientific). Amplification of spike gene from cDNA was performed using nested PCR and sequenced using Sanger sequencing (Genewiz). Multiple clones from limiting dilution nested PCR were sequenced to confirm the dominant mutants in the pool of the resulting progeny viruses and a percentage of their prevalence was calculated from total number sequenced. For passage 4, 9 and 12, a total of 20, 10 and 10 clones were sequenced respectively to confirm the mutations.

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