Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

Flow cytometry based cell surface SARS-CoV-1/CoV-2 spike binding assay Binding of mAbs/sera to the HEK293T cell-surface expressed SARS-CoV-1 and SARS-CoV-2 spikes was performed as described previously [18]. Briefly, HEK293T cells were transfected with plasmids encoding full-length SARS-CoV-1 or SARS-CoV-2 spikes and incubated for 36-48 hours at 37 °C. Post incubation cells were trypsinization to prepare single cell suspension and were distributed into 96-well plates. 50ul/well of 3-fold serial titrations of mAbs starting at 10 μg/mL or serum samples starting at 1:30 dilution were added to transfected cells. The Abs were incubated with cells for 1 hr on ice. The plates were washed twice in FACS buffer (1X PBS, 2% FBS, 1mM EDTA) and stained with 50ul/well of 1:200 dilution of R-phycoerythrin (PE)-conjugated mouse anti-human IgG Fc antibody (SouthernBiotech) and 1:1000 dilution of Zombie-NIR viability dye (BioLegend). After another two washes, stained cells were analyzed using flow cytometry (BD Lyrics cytometers), and the binding data were generated by calculating the percent (%) PE-positive cells for antigen binding using FlowJo 10 software. CR3022, a SARS-CoV-2 spike binding antibody, and unrelated antibody, DEN3, were respectively positive and negative controls for the assay.
Protein expression and purification To express the soluble S ectodomain protein SARS-CoV-1, SARS-CoV-2 and their truncated protein versions, protein-encoding plasmids were transfected into FreeStyle293F cells (Thermo Fisher) at a density of approximately 1 million cells/mL. For large scale production, we mixed 350ug plasmids with 16 mL transfectagro™ (Corning) in a conical tube and filtered with 0.22um Steriflip™ Sterile Disposable Vacuum Filter Units (MilliporeSigma™). In another conical tube, we added 1.8mL 40K PEI (1 mg/mL) into 16 mL transfectagro™ and mixed briefly. The premixed 40K PEI-transfectagro™ solution was gently poured into the filtered plasmid solution. The solution was thoroughly mixed by inverting the tube several times. The mixture rested at room temperature for 30 mins and was poured into 1L FreeStyle293F cell culture. After 5 days, the cells were removed from the supernatant by centrifuging at 3500 rpm for 15mins. The supernatant was filtered in a glass bottle with the 0.22um membrane and kept in 4-degree storage before loading into the columns. The His-tagged proteins were purified with the HisPur Ni-NTA Resin (Thermo Fisher). To eliminate the nonspecific binding proteins, each column was washed with at least 3 bed volumes of wash buffer (25 mM Imidazole, pH 7.4). To elute the purified proteins from the column, we loaded 25mL of the elution buffer (250mM Imidazole, pH 7.4) at slow gravity speed (~4 sec/drop). By using Amicon tubes, we buffer exchanged the solution with PBS and concentrated the proteins. The proteins were further purified by size-exclusion chromatography using Superdex 200 (GE Healthcare). The selected fractions were pooled and concentrated again for the further use.
Recombinant Protein ELISAs 6x-His tag monoclonal antibody (Invitrogen, UA280087) was coated onto high-binding 96-well plates (Corning, 3690) at 2 µg/mL overnight at 4 ℃. After washing, plates were blocked with 3% BSA in PBS for 1h. Then his-tag recombinant RBD and S protein were captured at 1µg/mL in 1% BSA and incubated for 1h at RT. After washing, serially diluted mAbs were added into wells and incubated for 1h at RT. Detection was measured with alkaline phosphatase-conjugated goat anti-human IgG Fcg (Jackson ImmunoResearch) at 1:1000 dilution for 1h. After the final wash, phosphatase substrate (Sigma-Aldrich) was added into wells. Absorption was measured at 405 nm.
12.5 ul of 6x-His tag monoclonal antibody (Invitrogen UA280087) were coated onto high binding plates at 2µg/mL overnight at 4℃. The plate was washed three times with 100 ul of 1xPBS/0.05% Tween. The recombinant RBD and S protein derived from SARS-CoV-1 and SARS-CoV-2 were diluted with 1xPBS/1%BSA to final concentrations of 2 µg/mL or 5 µg/mL and then coated onto the plate and incubated at RT for 2h. The plate was washed three times with 100 ul of 1xXPBS/0.05% Tween and then blocked with 100 ul of 3% BSA at RT for 2 hours and then the BSA was removed. Either serially diluted plasma samples or isolated mAbs were added onto the plate and incubated for 1.5 hours at RT. Wells were then incubated with secondary anti-human IgG Fcγ antibody diluted at 1:2000 and incubated at RT for 1 hour. The plate was then washed three times with 100 µL of 1xPBS/0.05% tween. 12.5uL of phosphatase substrate (SIGMA-ALDRICH S0942) were added to the plate and optical density (OD) was measured at 405 nm. Plasma or mAbs were tested in duplicate or triplicate wells. Non-linear regression curves were analyzed using Prism 8 software to calculate IC50 values.
Isolation of SARS-2 S-specific mAbs Sorting of antigen-specific memory B cells was performed as previously described [19,20]. The process was adapted for high-throughput such that each step could be performed in a 96-well format. Fluorescent-labeled antibodies recognizing cell surface markers were purchased from BD Biosciences. AVI-tagged SARS-2 S and RBD proteins were produced, purified, labeled with biotin (Avidity), and coupled to streptavidin-AF647, streptavidin-AF488 (Thermo Fisher), and streptavidin-BV421 (BD Biosciences), as previously described [20] at 2:1 and 4:1 molecular ratio respectively 30 min prior to staining. Cells were first labeled with antibodies for surface markers together with probes (200 nM final) for 30min in sort buffer (PBS 1% FBS, 2.5M EDTA, 25mM Hepes) on ice. Cells were then stained with the Live/Dead Fixable Aqua Dead Cell Stain (Thermo Fisher) for 15 minutes on ice according to the manufacturer's instructions. Single antigen-specific (S+ and RBD+) memory B cells (CD3-CD4-CD8-CD14-CD19+IgD-IgG+) were sorted into individual empty wells of a 96-well plate using a BD FACSAria Fusion sorter. Plates were immediately sealed and stored at-80°C. cDNA was generated from cells sorted using Superscript IV Reverse Transcriptase (Thermo Fisher), dNTPs (Thermo Fisher), random hexamers (Gene Link) and Ig gene-specific primers in a lysis buffer containing Igepal (Sigma), DTT and RNAseOUT (Thermo Fisher). Nested PCR amplification of heavyand light-chain variable regions was performed using Hot Start DNA Polymerases (Qiagen, Thermo Fisher), and previously described primer sets [21,22]. Second round PCR primers were modified to include additional nucleotides overlapping with the expression vectors. PCR efficiency was assessed using 96w E-gels (Thermo Fisher). Paired wells picked individually, re-arrayed into new 96w plates and cloned in-frame into expression vectors encoding the human IgG1, Ig kappa or Ig lambda constant domains using the Gibson Assembly Enzyme mix (New England BioLabs) according to manufacturer instructions. Ligation reactions were transformed into DH5a competent E-coli, transferred into 1mL Plasmid+ media (Thomson Instrument Company) supplemented with antibiotic and grown overnight at 37C under agitation. The next day the cultures were used to inoculate duplicate cultures before being lysed for plasmid DNA extraction using NucleoSpin 96 miniprep kit (Macherey-Nagel, Takara). Cloned heavy-and light-chain variable regions were sequenced (Genewiz) and subsequently analyzed using the IMGT (International ImMunoGeneTics Information System, www.imgt.org) V-quest webserver [23].
Flow cytometry-based cell surface ACE2 binding inhibition assay. Monoclonal antibody inhibition of SARS-CoV-2 S or RBD binding to cell surface hACE2 was performed by flow cytometry as follows. Purified mAbs were mixed with biotinylated SARS-CoV-2 S or RBD in the molar ratio of 4:1 on ice for 1h. In the meantime, HeLa-ACE2 cells were washed once with DPBS then detached by incubation with DPBS supplemented with 5 mM EDTA. The detached HeLa-ACE2 cells were washed and resuspended with FACS buffer (2% FBS and 1 mM EDTA in DPBS). 0.5 million Hela-ACE2 cells were added to mAb/S or RBD mixture and incubated at 4°C for 0.5 h. HeLa-ACE2 cells were then washed once with FACS buffer, resuspended FACS buffer with 1 µg/mL streptavidin-AF647 (Thermo, S21374) and incubated for another 0.5 h. After washing, HeLa-ACE2 cells were resuspended in FACS buffer in the presence of 2 µg/mL propidium iodide (Sigma, P4170-100MG) for live/dead staining. HeLa and HeLa-ACE2 cells stained with SARS-CoV-2 S or RBD alone were used as background and positive control separately. The AF647 mean fluorescence intensity (MFI) was determined from the gate of singlet and PI negative cells. The percentage of ACE2 binding inhibition was calculated using the following equation.
Antibody expression and purification Antibodies HC and LC constructs were transiently expressed with the Expi293 Expression System (Thermo fisher). After 4 days, 24-deep well culture supernatants were harvested to be directly tested for binding and neutralization. Selected mAbs showing neutralizing activity in the HTP screening were reexpressed in small to medium scale cultures using individual colonies plasmid DNA, and IgG purified on Protein A sepharose (GE Healthcare).
Epitope binning by bio-layer interferometry Neutralizing and non-neutralizing mAbs were binned into epitope specificities using an Octet RED384 system. 50-100 nM of HIS-tagged S or RBD protein antigens were captured using anti-Penta-HIS biosensors (18-5120, Molecular Devices). After antigen loading for 5 min, a saturating concentration of mAb (100 µg/mL) was added for 10 min. Competing concentrations of mAbs (25 µg/mL) were then added for 5 min to measure binding in the presence of saturating antibodies. All incubation steps were performed in 1x PBS.
Surface Plasmon Resonance Methods SPR measurements were collected using a Biacore 8K instrument at 25°C. All experiments were carried out with a flow rate of 30 µL/min in a mobile phase of HBS-EP+ [0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.0005% (v/v) Surfactant P20]. Anti-Human IgG (Fc) antibody (Cytiva) was immobilized to a density of ~7000-10000 RU via standard NHS/EDC coupling to a Series S CM-5 (Cytiva) sensor chip. A reference surface was generated through the same method.
For conventional kinetic/dose-response, listed antibodies were captured to ~50-100 RU via Fc-capture on the active flow cell prior to analyte injection. A concentration series of SARS-CoV-2 RBD was injected across the antibody and control surface for 2 min, followed by a 5 min dissociation phase using a multicycle method. Regeneration of the surface in between injections of SARS-CoV-2 RBD was achieved with a single, 120 s injection of 3M MgCl2. Kinetic analysis of each reference subtracted injection series was performed using the BIAEvaluation software (Cytiva). All sensorgram series were fit to a 1:1 (Langmuir) binding model of interaction.
A SPR assay was also used to assess the competition between SARS-CoV-2 RBD and ACE2 for binding to CC12.1 [24,25]. CC12.1 was captured to the surface of 3 flow cells to ~100 RU via Fc-capture. SARS-CoV-2 RBD was injected to each flow cell at a concentration of 50 nM to establish a basal level of SARS-CoV-2 RBD binding. This concentration was held constant for the competition experiments, which were carried out by varying the ACE2 concentration over eight points from 800 to 6.25 nM. To calculate residual SARS-CoV-2 RBD binding, the sensorgram responding to the corresponding ACE2 injection alone was subtracted from the SARS-CoV-2 RBD plus ACE2 injection series. The average response for the 5 s preceding the injection stop was plotted against the concentration of ACE2 and fit to a doseresponse inhibition curve by nonlinear regression [log(inhibitor) vs. response -variable slope (4 parameters)] using GraphPad Prism. Regeneration between injections was carried out as noted above.
Animal Study SARS-CoV-2 infection of 8-week old Syrian hamsters was achieved through the intranasal instillation of 10 6 total PFU per animal in 100 µl of PBS. Animals weights were obtained during the study as a measure of disease progression. Treatment groups included the intraperitoneal injection of varying doses of monoclonal antibody. After 12-h, serum was obtained to quantify mAb titers and animals were infected as described above. At day-5 post-infection, lungs were harvested for analysis. Research protocol was approved and performed in accordance with Scripps Research IACUC Protocol #20-0003. Viral load measurements Viral RNA was isolated from lung tissue and subsequently amplified and quantified in a RT-qPCR reaction. Lung tissue was extracted at day 5 post infection. The lung tissue was divided into sections approximately 100-300mg in size. Samples were placed in 1mL of TRIzol-LS reagent (Invitrogen). Samples for virus load were then subjected to tissue homogenization using disposable pestles in 15mL conical tubes (Corning). Tissue homogenates were then spun down to remove any remaining cellular debris and the supernatant was added to a RNA purification column (Qiagen). Purified RNA was eluted in 80uL of DNase-, RNase-, endotoxin-free molecular biology grade water (Millipore) and quantified using a nanodrop (Thermo Fisher). RNA was then subjected to reverse transcription and quantitative PCR using the CDC's N1 primer sets (Forward 5'-GAC CCC AAA ATC AGC GAA AT-3'; Reverse 5'-TCT GGT TAC TGC CAG TTG AAT CTG-3') and a double-quenched (ZEN/Iowa Black FQ) and fluorescently labeled (FAM) probe (5'-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3') (Integrated DNA Technologies) on an BioRad CFX96 Real-Time instrument. For quantification, a standard curve was generated by diluting 1 × 1010 RNA copies SARS-CoV-2 genome/mL, 10-fold in water (Virapur). Every run utilized eight, tenfold serial dilutions of the standard. SARS-CoV-2-positive and -negative samples were included.

CoV-2 FL
CoV-1-FL         Fig. S7A: Monoclonal antibodies were evaluated for epitope competition using an Octet RED384 platform. His-tagged RBD was captured using an anti-Penta-HIS biosensor and indicated mAbs at a concentration of 100 µg/ml are first incubated for 10 min followed by incubation with 25 µg/ml of competing antibodies for 5 min.   Purified mAbs were mixed with biotinylated SARS-CoV-2 S or RBD proteins at a molar ratio of 4:1 before adding to HeLa-ACE2 cells. Streptavidin-AF647 was used to detect the relative intensity of cell surface ACE2 binding biotinylated SARS-CoV-2 S or RBD proteins. SARS-CoV-2 S or RBD-AF647 mean fluorescence intensity (MFI) was determined from the gate of singlet and PI negative cells. CR3022 and Den3 were used as negative controls. HeLa and HeLa-ACE2 cells stained with SARS-CoV-2 S or RBD alone were used as background and positive control separately.    Purified mAbs were mixed with biotinylated SARS-CoV-2 S or RBD proteins at a molar ratio of 4:1 before adding to HeLa-ACE2 cells. Streptavidin-AF647 was used to detect the relative intensity of cell surface ACE2 binding biotinylated SARS-CoV-2 S or RBD proteins. SARS-CoV-2 S or RBD-AF647 mean fluorescence intensity (MFI) was determined from the gate of singlet and PI negative cells. CR3022 and Den3 were used as negative control. HeLa and HeLa-ACE2 cells stained with SARS-CoV-2 S or RBD alone were used as background and positive control separately.