Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rino Rappuoli (rino.r.rappuoli@gsk.com).

Materials availability

Reasonable amounts of antibodies will be made available by the Lead Contact upon request under a Material Transfer Agreement (MTA) for non-commercial usage.

Data and code availability

Nucleotide and amino acidic sequences of all SARS-CoV-2-neutralizing antibodies were deposited in the Italian patent applications n. 102020000015754 filed on June 30th 2020 and 102020000018955 filed on August 3rd 2020. The accession number for the nucleotide sequences of all SARS-CoV-2-neutralizing antibodies reported in this paper is GenBank: MW_598287 - MW_598314.

Enrollment of SARS-COV-2 convalescent donors and human sample collection

This work results from a collaboration with the National Institute for Infectious Diseases, IRCCS – Lazzaro Spallanzani Rome (IT) and Azienda Ospedaliera Universitaria Senese, Siena (IT) that provided samples from SARS-CoV-2 convalescent donors, of both sexes, who gave their written consent. The study was approved by local ethics committees (Parere 18_2020 in Rome and Parere 17065 in Siena) and conducted according to good clinical practice in accordance with the declaration of Helsinki (European Council 2001, US Code of Federal Regulations, ICH 1997). This study was unblinded and not randomized.

Single cell sorting of SARS-CoV-2 S-protein⁺ memory B cells from COVID-19 convalescent donors

Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMC were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) in 100 μL final volume diluted 1:500 at room temperature RT. After 20 min incubation cells were washed with PBS and unspecific bindings were saturated with 50 μL of 20% normal rabbit serum (Life technologies) in PBS . Following 20 min incubation at 4°C cells were washed with PBS and stained with SARS-CoV-2 S-protein labeled with Strep-Tactin®XT DY-488 (iba-lifesciences cat# 2-1562-050) for 30 min at 4°C. After incubation the following staining mix was used CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min. Stained MBCs were single cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described (Huang et al., 2013).

Expression and purification of SARS-CoV-2 S-protein prefusion trimer and receptor binding domain

Plasmid encoding SARS-CoV-2 S-2P construct (Wrapp et al., 2020) and S-protein RBD (generously provided by Jason S. McLellan) were transiently transfected at 1 μg/mL culture into Expi293F cells (Thermo Fisher) using ExpiFectamine 293 Reagent. Cells were grown for six days at 37°C with 8% CO₂ shaking 125 rpm according to the manufacturer’s protocol (Thermo Fisher); ExpiFectamine 293 Transfection Enhancers 1 and 2 were added 16 to 18 h post-transfection to boost transfection, cell viability, and protein expression. Cell cultures were harvested three and six days after transfection. Cells were separated from the medium by centrifugation (1,100 g for 10 min at 24°C). Collected supernatants were then pooled and clarified by centrifugation (3,000 g for 15 min at 4°C) followed by filtration through a 0.45 μm filter. Chromatography was conducted at room temperature using the ÄKTA go purification system from GE Healthcare Life Sciences. Expressed proteins were purified by using an immobilized metal affinity chromatography (FF Crude) followed by dialysis into final buffer. Specifically, the filtrated culture supernatant was purified with a 5 mL HisTrap FF Crude column (GE Healthcare Life Sciences) previously equilibrated in Buffer A (20 mM NaH₂PO₄, 500 mM NaCl + 30 mM imidazol pH 7.4).

The flow rate for all steps of the HisTrap FF Crude column was 5 mL/min. The culture supernatant of spike and RBD cell culture was applied to a single 5 mL HisTrap FF Crude column. The column was washed in Buffer A for 4 column volumes (CV) with the all 4 CV collected as the column wash. Recombinant proteins were eluted from the column applying a first step elution of 4 CV of 50% Buffer B (20 mM NaH2PO4, 500 mM NaCl + 500 mM imidazol pH 7.4) and a second step elution of 2 CV of 100% Buffer B. Elution steps were collected in 1 fractions of 1 mL each. Eluted fractions were analyzed by SDS-PAGE and appropriate fractions containing recombinant proteins were pooled. Final pools were dialyzed against phosphate buffered saline (PBS) pH 7.4 using Slide-A-Lyzer G2 Dialysis Cassette 3.5K (Thermo Scientific) overnight at 4°C. The dialysis buffer used was at least 200 times the volume of the sample.

The final protein concentration was determined by measuring the A520 using the Pierce BCA protein assay kit (Thermo Scientific). Final protein was dispensed in aliquots of 0.5 mL each and stored at −80°C.

ELISA assay with S1 and S2 subunits of SARS-CoV-2 S-protein

The presence of S1- and S2-binding antibodies in culture supernatants of monoclonal S-protein-specific memory B cells was assessed by means of an ELISA implemented with the use of a commercial kit (ELISA Starter Accessory Kit, Catalogue No. E101; Bethyl Laboratories). Briefly, 384-well flat-bottom microtiter plates (384 well plates, Microplate Clear, Greiner Bio-one) were coated with 25 μL/well of antigen (1:1 mix of S1 and S2 subunits, 1 μg/mL each; The Native Antigen Company, Oxford, United Kingdom) diluted in coating buffer (0.05 M carbonate-bicarbonate solution, pH 9.6), and incubated overnight at 4°C. The plates were then washed three times with 100 μL/well washing buffer (50 mM Tris Buffered Saline (TBS) pH 8.0, 0.05% Tween-20) and saturated with 50 μL/well blocking buffer containing Bovine Serum Albumin (BSA) (50 mM TBS pH 8.0, 1% BSA, 0.05% Tween-20) for 1 h (h) at 37°C. After further washing, samples diluted 1:5 in blocking buffer were added to the plate. Blocking buffer was used as a blank. After an incubation of 1 h at 37°C, plates were washed and incubated with 25 μL/well secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-human IgG-Fc Fragment polyclonal antibody, diluted 1:10,000 in blocking buffer, Catalogue No. A80-104P; (Bethyl Laboratories) for 1 h at 37°C. After three washes, 25 μL/well TMB One Component HRP Microwell Substrate (Bethyl Laboratories) was added and incubated 10–15 min at RT in the dark. Color development was terminated by addition of 25 μL/well 0.2 M H₂SO₄. Absorbance was measured at 450 nm in a Varioskan Lux microplate reader (Thermo Fisher Scientific). Plasma from COVID-19 convalescent donors (Andreano et al., 2020) and unrelated plasma were used as positive and negative control respectively. The threshold for sample positivity was set at twice the optical density (OD) of the blank.

ELISA assay with SARS-CoV-2 S-protein prefusion trimer and S1 – S2 subunits

ELISA assay was used to detect SARS-CoV-2 S-protein specific mAbs and to screen plasma from SARS-CoV-2 convalescent donors. 384-well plates (384 well plates, microplate clear; Greiner Bio-one) were coated with 3 μg/mL of streptavidin (Thermo Fisher) diluted in coating buffer (0.05 M carbonate-bicarbonate solution, pH 9.6) and incubated at RT overnight. Plates were then coated with SARS-CoV-2 S-protein, S1 or S2 domains at 3 μg/mL and incubated for 1 h at RT. 50 μL/well of saturation buffer (PBS/BSA 1%) was used to saturate unspecific binding and plates were incubated at 37°C for 1 h without CO₂. For the first round of screening, supernatants were diluted 1:5 in PBS/BSA 1%/Tween20 0.05% in 25 μL/well final volume and incubated for 1 h at 37°C without CO₂. For purified antibodies, and to assess EC₅₀, mAbs were tested at a starting concentration of 5 μg/mL and diluted step 1:2 in PBS/BSA 1%/Tween20 0.05% in 25 μL/well final volume for 1 h at 37°C without CO₂. 25 μL/well of alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich) and IgA (Southern Biotech) were used as secondary antibodies. Wells were washed three times between each step with PBS/BSA 1%/Tween20 0.05%. pNPP (p-nitrophenyl phosphate) (Sigma-Aldrich) was used as soluble substrate to detect SARS-CoV-2 S-protein, S1 or S2 specific mAbs and the final reaction was measured by using the Varioskan Lux Reader (Thermo Fisher Scientific) at a wavelength of 405 nm. Plasma from COVID-19 convalescent donors (Andreano et al., 2020) and unrelated plasma were used as positive and negative control respectively. Samples were considered as positive if OD at 405 nm (OD₄₀₅) was twice the blank.

SARS-CoV-2 virus and cell infection

African green monkey kidney cell line Vero E6 cells (American Type Culture Collection [ATCC] #CRL-1586) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) - high glucose (Euroclone, Pero, Italy) supplemented with 2 mM L- Glutamine (Lonza, Milano, Italy), penicillin (100 U/mL) - streptomycin (100 μg/mL) mixture (Lonza, Milano, Italy) and 10% Foetal Bovine Serum (FBS) (Euroclone, Pero, Italy). Cells were maintained at 37°C, in a 5% CO₂ humidified environment and passaged every 3-4 days.

Wild type SARS-CoV-2 (SARS-CoV-2/INMI1-Isolate/2020/Italy: MT066156), D614G (SARS-CoV-2/human/ITA/INMI4/2020, clade GR, D614G (S): MT527178) and B.1.1.7 (INMI-118 GISAID accession number EPI_ISL_736997) viruses were purchased from the European Virus Archive goes Global (EVAg, Spallanzani Institute, Rome)or received from the Spallanzani Institute, Rome. For virus propagation, sub-confluent Vero E6 cell monolayers were prepared in T175 flasks (Sarstedt) containing supplemented D-MEM high glucose medium. For titration and neutralization tests of SARS-CoV-2, Vero E6 were seeded in 96-well plates (Sarstedt) at a density of 1.5 × 10⁴ cells/well the day before the assay.

Neutralization of Binding (NoB) Assay

To study the binding of the SARS-CoV-2 S-protein to cell-surface receptor(s) we developed an assay to assess recombinant S-protein specific binding to target cells and neutralization thereof. To this aim the stabilized S-protein was coupled to Streptavidin-PE (eBioscience # 12-4317-87, Thermo Fisher) for 1 h at RT and then incubated with Vero E6 cells. Binding was assessed by flow cytometry. The stabilized S-protein bound Vero E6 cells with high affinity (data not shown). To assess the content of neutralizing antibodies in sera or in B cell culture supernatants, two microliters of SARS-CoV-2 Spike-Streptavidin-PE at 5 - 10 μg/mL in PBS-5%FCS were mixed with two microliters of various dilutions of sera or B cell culture supernatants in U bottom 96-well plates. After incubation at 37°C for 1 h, 30 × 10³ Vero E6 cells suspended in two microliters of PBS 5% FCS were added and incubated for additional 45 min at 4°C. Non-bound protein and antibodies were removed and cell-bound PE-fluorescence was analyzed with a FACS Canto II flow cytometer (Becton Dickinson). Data were analyzed using the FlowJo data analysis software package (TreeStar, USA). The specific neutralization was calculated as follows: NoB (%) = 1 – (Sample MFI value – background MFI value) / (Negative Control MFI value – background MFI value). Plasma from COVID-19 convalescent donors (Andreano et al., 2020) and unrelated plasma were used as positive and negative control respectively.

Single cell RT-PCR and Ig gene amplification

From the original 384-well sorting plate, 5 μL of cell lysate was used to perform RT-PCR. Total RNA from single cells was reverse transcribed in 25 μL of reaction volume composed by 1 μL of random hexamer primers (50 ng/μL), 1 μL of dNTP-Mix (10 mM), 2 μL 0.1 M DTT, 40 U/μL RNase OUT, MgCl₂ (25 mM), 5x FS buffer and Superscript IV reverse transcriptase (Invitrogen). Final volume was reached by adding nuclease-free water (DEPC). Reverse transcription (RT) reaction was performed at 42°C/10’, 25°C/10’, 50°C/60’ and 94°/5¢. Heavy (VH) and light (VL) chain amplicons were obtained via two rounds of PCR. All PCR reactions were performed in a nuclease-free water (DEPC) in a total volume of 25 μL/well. Briefly, 4 μL of cDNA were used for the first round of PCR (PCRI). PCRI-master mix contained 10 μM of VH and 10 μM VL primer-mix, 10mM dNTP mix, 0.125 μL of Kapa Long Range Polymerase (Sigma), 1.5 μL MgCl2 and 5 μL of 5x Kapa Long Range Buffer. PCRI reaction was performed at 95°/3¢, 5 cycles at 95°C/30’’, 57°C/30’’, 72°C/30’’ and 30 cycles at 95°C/30’’, 60°C/30’’, 72°C/30’’ and a final extension of 72°/2’. All nested PCR reactions (PCRII) were performed using 3.5 μL of unpurified PCRI product using the same cycle conditions. PCRII products were then purified by Millipore MultiScreen® PCRμ96 plate according to manufacture instructions. Samples were eluted with 30 μL nuclease-free water (DEPC) into 96-well plates and quantify by.

Cloning of variable region genes and recombinant antibody expression in transcriptionally active PCR

Vector digestions were carried out with the respective restriction enzymes AgeI, SalI and Xho as previously described (Tiller et al., 2008, Wardemann and Busse, 2019). Briefly, 75 ng of IgH, Igλ and Igκ purified PCRII products were ligated by using the Gibson Assembly NEB into 25 ng of respective human Igγ1, Igκ and Igλ expression vectors. The reaction was performed into 5 μL of total volume. Ligation product was 10-fold diluted in nuclease-free water (DEPC) and used as template for transcriptionally active PCR (TAP) reaction which allowed the direct use of linear DNA fragments for in vitro expression. The entire process consists of one PCR amplification step, using primers to attach functional promoter (human CMV) and terminator sequences (SV40) onto the fragment PCRII products. TAP reaction was performed in a total volume of 25 μL using 5 μL of Q5 polymerase (NEB), 5 μL of GC Enhancer (NEB), 5 μL of 5X buffer,10 mM dNTPs, 0.125 μL of forward/reverse primers and 3 μL of ligation product. TAP reaction was performed by using the following cycles: 98°/2’, 35 cycles 98°/10’’, 61°/20’’, 72°/1’ and 72°/5¢ as final extention step. TAP products were purified under the same PCRII conditions, quantified by Qubit Fluorometric Quantitation assay (Invitrogen) and used for transient transfection in Expi293F cell line using manufacturing instructions.

Flask expression and purification of human monoclonal antibodies

Expi293F cells (Thermo Fisher) were transiently transfected with plasmids carrying the antibody heavy chain and the light chains with a 1:2 ratio. Cells were grown for six days at 37°C with 8% CO₂ shaking at 125 rpm according to the manufacturer’s protocol (Thermo Fisher); ExpiFectamine 293 transfection enhancers 1 and 2 were added 16 to 18 h post-transfection to boost cell viability and protein expression. Cell cultures were harvested three and six days after transfection. Cells were separated from the medium by centrifugation (1,100 g for 10 min at 24°C). Supernatants collected were then pooled and clarified by centrifugation (3000 g for 15 min, 4°C) followed by filtration through a 0.45 μm filter. Chromatography was conducted at room temperature using the ÄKTA go purification system from GE Healthcare Life Sciences. Affinity chromatography was used to purify expressed monoclonal antibodies using an immobilized protein G column able to bind to Fc region. Specifically, filtrated culture supernatants were purified with a 1 mL HiTrap Protein G HP column (GE Healthcare Life Sciences) previously equilibrated in Buffer A (0.02 M NaH₂PO₄ pH 7). The flow rate for all steps of the HiTrap Protein G HP column was 1 mL/min. The culture supernatant for every monoclonal antibody cell culture was applied to a single 1 mL HiTrap Protein G HP column. The column was equilibrated in Buffer A for at least 6 column volumes (CV) which was collected as column wash. Each monoclonal antibody was eluted from the column applying a step elution of 6 CV of Buffer B (0.1 M glycine-HCl, pH 2.7). Elution steps were collected in 1 fractions of 1 mL each. Eluted fractions were analyzed by non-reducing SDS-PAGE and fractions showing the presence of IgG were pooled together. Final pools was dialyzed in PBS buffer pH 7.4 using Slide-A-Lyzer G2 Dialysis Cassette 3.5K (Thermo Scientific) overnight at 4°C. The dialysis buffer used was at least 200 times the volume of the sample. For each antibody purified the concentration was determined by measuring the A520 using Pierce BCA Protein Assay Kit (Thermo Scientific). All the purified antibodies were aliquoted and stored at −80°C.

Viral propagation and titration

The SARS-CoV-2 virus was propagated in Vero E6 cells cultured in DMEM high Glucose supplemented with 2% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin. Cells were seeded at a density of 1x10⁶ cells/mL in T175 flasks and incubated at 37°C, 5% CO₂ for 18 - 20 h. The sub-confluent cell monolayer was then washed twice with sterile Dulbecco’s PBS (DPBS). Cells were inoculated with 3,5 mL of the virus properly diluted in DMEM 2% FBS at a multiplicity of infection (MOI) of 0.001, and incubated for 1 h at 37°C in a humidified environment with 5% CO₂. At the end of the incubation, 50 mL of DMEM 2% FBS were added to the flasks. The infected cultures were incubated at 37°C, 5% CO₂ and monitored daily until approximately 80%–90% of the cells exhibited cytopathic effect (CPE). Culture supernatants were then collected, centrifuged at 4°C at 1,600 rpm for 8 min to allow removal of cell debris, aliquoted and stored at −80°C as the harvested viral stock. Viral titers were determined in confluent monolayers of Vero E6 cells seeded in 96-well plates using a 50% tissue culture infectious dose assay (TCID₅₀). Cells were infected with serial 1:10 dilutions (from 10⁻¹ to 10⁻¹¹) of the virus and incubated at 37°C, in a humidified atmosphere with 5% CO₂. Plates were monitored daily for the presence of SARS-CoV-2 induced CPE for 4 days using an inverted optical microscope. The virus titer was estimated according to Spearman-Karber formula (Kundi, 1999) and defined as the reciprocal of the highest viral dilution leading to at least 50% CPE in inoculated wells.

SARS-CoV-2 authentic virus neutralization assay

All SARS-CoV-2 authentic virus neutralization assays were performed in the biosafety level 3 (BSL3) laboratories at Toscana Life Sciences in Siena (Italy) and Vismederi Srl, Siena (Italy). BSL3 laboratories are approved by a Certified Biosafety Professional and are inspected every year by local authorities. The neutralization activity of culture supernatants from monoclonal was evaluated using a CPE-based assay as previously described (Manenti et al., 2020). S-protein-specific memory B cells produced antibodies were initially evaluated by means of a qualitative live-virus based neutralization assay against a one-point dilution of the samples. Supernatants were mixed in a 1:3 ratio with a SARS-CoV-2 viral solution containing 25 TCID₅₀ of virus (final volume: 30 μL). After 1 h incubation at 37°C, 5% CO₂, 25 μL of each virus-supernatant mixture was added to the wells of a 96-well plate containing a sub-confluent Vero E6 cell monolayer. Following a 2 h incubation at 37°C, the virus-serum mixture was removed and 100 μl of DMEM 2% FBS were added to each well. Plates were incubated for 3 days at 37°C in a humidified environment with 5% CO₂, then examined for CPE by means of an inverted optical microscope. Absence or presence of CPE was defined by comparison of each well with the positive control (plasma sample showing high neutralizing activity of SARS-CoV-2 in infected Vero E6 cells (Andreano et al., 2020) and negative control (human serum sample negative for SARS-CoV-2 in ELISA and neutralization assays). Following expression as full-length IgG1 recombinant antibodies were quantitatively tested for their neutralization potency against both the wild type, D614G variant and the B.1.1.7 emerging variants. The assay was performed as previously described but using a viral titer of 100 TCID₅₀. Antibodies were prepared at a starting concentration of 20 μg/mL and diluted step 1:2. Technical triplicates were performed for each experiment.

Production and titration of SARS-CoV-2 pseudotyped lentiviral reporter particles

Pseudotype stocks were prepared by FuGENE-HD (Promega) co-transfection of HEK293T/17 with SARS-CoV-2 spike pcDNA3.1 + expression plasmid, HIV gag-pol p8.91 plasmid and firefly luciferase expressing plasmid pCSFLW in a 1:0.8:1.2 ratio. 2 × 10⁶ cells/cm² were plated 24 h prior to transfection in 10cm cell culture dishes. 48 and 72 h post transfection, pseudotype-containing culture medium was harvested and filtered thought a 0.45um syringe filter to clear cell debris. Aliquots were stored at −80°C. Titration assays were performed by transduction of HEK293T/17 cells pre-transfected with ACE2 and TMPRRS2 plasmids to calculate the viral titer and infectious dose (PV input) for neutralization assays. SARS-CoV-2 D614G pseudotype was produced using the same procedure as described above. SARS-1 pseudotype was produced in a 1:0.5:0.8 ratio. MERS-pseudotype was produced as previously described (Grehan et al., 2015).

SARS-CoV-2 pseudotyped lentivirus neutralization assay

The potency of the neutralizing mAbs was assessed using lentiviral particles expressing SARS-CoV-2 spike protein on their surface and containing firefly luciferase as marker gene for detection of infection. Briefly, 2 × 10⁶ HEK293T cells were pre-transfected in a 10 cm dish the day before the neutralization assay with ACE2 and TMPRSS2 plasmids in order to be used as optimal target cells for SARS-CoV-2 PV entry. In a 96-well plate mAbs were 2-fold serially diluted in duplicate in culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) starting at 20 μg/mL in a total volume of 100 μL. 1x10⁶ RLU of SARS-CoV-2 pseudotyped lentiviral particles were added to each well and incubated at 37°C for 1 h. Each plate included PV plus cell only (virus control) and cells only (background control). 1 × 10⁴ pre-transfected HEK293T cells suspended in 50 μL complete media were added per well and incubated for 48 h at 37°C and 5% CO2. Firefly luciferase activity (luminescence) was measured using the Bright-Glo assay system with a GloMax luminometer (Promega, UK). The raw Relative Luminescence Unit (RLU) data points were converted to a percentage neutralization value, whereby 100% neutralization equals the mean cell only RLU value control and 0% neutralization equals the mean PV only RLU value control. The normalized data was then plotted using Prism 8 (GraphPad) on a neutralization percentage scale and a NT50 value calculated, using the non-linear regression analysis. Plasma from COVID-19 convalescent donors showing neutralization activity against SARS-CoV-2 (Andreano et al., 2020) were also assessed in this assay.

Characterization of SARS-CoV-2 RBD-Antibodies binding by Flow cytometry

Flow cytometry analysis was performed to define antibodies interaction with S-protein-receptor-binding domain (RBD). Briefly, APEX Antibody Labeling Kits (Invitrogen) was used to conjugate 20 μg of selected antibodies to Alexa fluor 647, according to the manufacturer instructions. To assess the ability of each antibody to bind the RBD domain, 1 mg of magnetic beads (Dynabeads His-Tag, Invitrogen) were coated with 70 μg of histidine tagged RBD, and then 20 μg/mL of each labeled antibody were incubated with 40 μg/mL of beads-bound RBD for 1 h on ice. Then, samples were washed with 200 μL of Phosphate-buffered saline (PBS), resuspended in 150 μL of PBS and assessed with a FACS Canto II flow cytometer (Becton Dickinson). Results were analyzed by FlowJo (version 10).

Flow Cytometry-Based S-protein Competition assay

Antibodies specificity to bind SARS-CoV-2 S-protein and their possible competition was analyzed performing a Flow cytometer-based assay. To this aim, 200 μg of stabilized histidine tagged S-protein were coated with 1 mg of magnetic beads (Dynabeads His-Tag, Invitrogen). 20 μg of each antibody were labeled with Alexa fluor 647 working with the APEX Antibody Labeling Kits (Invitrogen). To test competitive binding profiles of the antibody panel selected, beads-bound S-protein (40 μg/mL) were pre-incubated with unlabeled antibodies (40 μg/mL) for 1 h on ice. Then, each set of the beads-antibody complexes were washed with PBS and separately incubated with each labeled antibody (20 μg/mL) for 1 h on ice. After incubation, the mix Beads-antibodies was washed, resuspended in 150 μL of PBS and analyzed using FACS Canto II flow cytometer (Becton Dickinson). Beads-bound and non-bound S-protein incubated with labeled antibodies were used as positive and negative control, respectively. Population gating and analysis was carried out using FlowJo (version 10).

Antigen-specific FcγR binding

Fluorescently coded microspheres were used to profile the ability of selected antibodies to interact with Fc receptors (Boudreau et al., 2020). The antigen of interest (SARS –CoV-2 S-protein RBD) was covalently coupled to different bead sets via primary amine conjugation. The beads were incubated with diluted antibody (diluted in PBS), allowing “on bead” affinity purification of antigen-specific antibodies. The bound antibodies were subsequently probed with tetramerized recombinant human FcγR2A and FcRN and analyzed using Luminex. The data is reported as the median fluorescence intensity of PE for a specific bead channel.

Antibody-dependent neutrophil phagocytosis

Antibody-dependent neutrophil phagocytosis (ADNP) assesses the ability of antibodies to induce the phagocytosis of antigen-coated targets by primary neutrophils. The assay was performed as previously described (Karsten et al., 2019, Boudreau et al., 2020). Briefly, fluorescent streptavidin-conjugated polystyrene beads were coupled to biotinylated SARS-CoV-2 Spike trimer. Diluted antibody (diluted in PBS) was added, and unbound antibodies were washed away. The antibody:bead complexes are added to primary neutrophils isolated from healthy blood donors using negative selection (StemCell EasySep Direct Human Neutrophil Isolation Kit), and phagocytosis was allowed to proceed for 1 h. The cells were then washed and fixed, and the extent of phagocytosis was measured by flow cytometry. The data is reported as a phagocytic score, which considers the proportion of effector cells that phagocytosed and the degree of phagocytosis. Each sample is run in biological duplicate using neutrophils isolated from two distinct donors. The mAb were tested for ADNP activity at a range of 30 μg/mL to 137.17 ng/mL.

Antibody-dependent NK cell activation

Antibody-dependent NK cell activation (ADNKA) assesses antigen-specific antibody-mediated NK cell activation against protein-coated plates. This assay was performed as previously described (Boudreau et al., 2020). Stabilized SARS-CoV-2 Spike trimer was used to coat ELISA plates, which were then washed and blocked. Diluted antibody (diluted in PBS) was added to the antigen coated plates, and unbound antibodies were washed away. NK cells, purified from healthy blood donor leukopaks using commercially available negative selection kits (StemCell EasySep Human NK Cell Isolation Kit) were added, and the levels of IFN-γ was measured after 5 h using flow cytometry. The data is reported as the percent of cells positive for IFN-γ. Each sample is tested with at least two different NK cell donors, with all samples tested with each donor. The monoclonal antibodies were tested for ADNKA activity at a range of 20 μg/mL to 9.1449 ng/mL.

Affinity evaluation of SARS-CoV-2 neutralizing antibodies

Anti-Human IgG Polyclonal Antibody (Southern Biotech 2040-01) was immobilized via amine group on two flow cells of a CM5 sensor chip. For the immobilization, anti-human IgG Ab diluted in 10mM Na acetate pH 5.0 at the concentration of 25 μg/mL was injected for 360 s over the dextran matrix, which had been previously activated with a mixture of 0.1M 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.4 M N-hydroxyl succinimide (NHS) for 420 s. After injection of the antibody, Ethanolamine 1M was injected to neutralize activated group. 10 μL/min flow rate was used during the whole procedure. Anti-SPIKE protein human mAbs were diluted in HBS-EP+ (HEPES 10 mM, NaCl 150 mM, EDTA 3.4 mM, 0.05% p20, pH 7.4) and injected for 120 s at 10 μL/min flow rate over one of the two flow cells containing the immobilized Anti-Human IgG Antibody, while running buffer (HBS-EP+) was injected over the other flow cell to be taken as blank. Dilution of each mAb was adjusted in order to have comparable levels of RU for each capture mAb. Following the capture of each mAb by the immobilized anti-human IgG antibody, different concentrations of SPIKE protein (20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL and 1 μg/mL in HBS-EP+) were injected over both the blank flow cell and the flow cell containing the captured mAb for 180 s at a flow rate of 80 μL/min. Dissociation was followed for 800 s, regeneration was achieved with a pulse (60 s) of Glycine pH 1.5. Kinetic rates and affinity constant of SPIKE protein binding to each mAb were calculated applying a 1:1 binding as fitting model using the Bia T200 evaluation software 3.1.

Autoreactivity screening test on HEp-2 Cells

The NOVA Lite HEp-2 ANA Kit (Inova Diagnostics) was used in accordance to the manufacturer’s instructions to test antibodies the autoreactivity of selected antibodies which were tested at a concentration of 100 μg/mL. Kit positive and negative controls were used at three different dilutions (1:1, 1:10 and 1:100). Images were acquired using a DMI3000 B microscope (Leica) and an exposure time of 300 ms, channel intensity of 2000 and a gamma of 2.

Genetic Analyses of SARS-CoV-2 S-protein specific nAbs

A custom pipeline was developed for the analyses of antibody sequences and the characterization of the immunoglobulin genes. Raw sequences were stored as ab1 file and transformed into fastaq using Biopython. The reads were then quality checked using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and a report was generated using MultiQC (https://multiqc.info/). The antibody leader sequence and the terminal part of the constant region were removed by trimming using Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic). This latter program was also used to scan and remove low-quality reads using a sliding-window parameter. Once sequences were recovered, germline gene assignment and annotation were perfromed with MiXCR (https://mixcr.readthedocs.io/en/master/index.html), using the single-read alignment parameters, and a CSV-formatted output was generated. Finally, the sequences retrieved from the antibodies described in this manuscript were compared to published neutralizing antibodies against SARS-CoV-2. For this purpose, the Coronavirus Antibody Database, CoV-AbDab (http://opig.stats.ox.ac.uk/webapps/covabdab/) was downloaded and the antibodies with reported neutralization activity against SARS-CoV-2 were extracted. Comparison analysis were performed in Python using NumPy (https://numpy.org/) and, Pandas (https://pandas.pydata.org/) while figures were produced using the Matplotlib tool (https://matplotlib.org/) and Seaborn (https://seaborn.pydata.org/).

Negative-stain electron microscopy

Complexes were formed by incubating SARS-2 CoV-GSAS-6P-Mut7 and respective fabs at a 1:3 (trimer to fab) molar ratio for 30 min at room temperature. After diluting to 0.03 mg/mL in 1X TBS pH 7.4, the samples were deposited on plasma-cleaned copper mesh grids and stained with 2% uranyl formate for 55 s. Automated data collection was made possible through the Leginon software (Suloway et al., 2005) and a FEI Tecnai Spirit (120keV, 56,000x mag) paired with a FEI Eagle (4k by 4k) CCD camera. Other details include a defocus value of −1.5 μm, a pixel size of 2.06 Å per pixel, and a dose of 25 e⁻/Ų. Raw micrographs were stored in the Appion database (Lander et al., 2009), particles were picked with DoGPicker (Voss et al., 2009), and 2D and 3D classification and refinements were performed in RELION 3.0 (Scheres, 2012). Map segmentation and model docking was done in UCSF Chimera (Pettersen et al., 2004).

Prophylactic and therapeutic passive transfer studies in golden Syrian hamsters

Six- to eight-month-old female Syrian hamsters were purchased from Charles River Laboratories and housed in microisolator units, allowed free access to food and water and cared for under U.S. Department of Agriculture (USDA) guidelines for laboratory animals. For the passive transfer prophylactic experiments, the day prior to SARS-CoV-2 infection six hamsters per group were intraperitoneally administered with 500 μL of a 4, 1 or 0.25 mg/kg dose of J08-MUT mAb. For the passive transfer therapeutic experiments, the day after SARS-CoV-2 infection six hamsters per group were intraperitoneally administered with 500 μL of a 4 mg/kg dose of J08-MUT mAb. Another two groups (n = 6/each) were administered with 500 μL of 4 mg/kg of the anti-influenza virus #1664 human mAb (manuscript in preparation) or PBS only to serve as human IgG1 isotype and mock control groups, respectively. The day after, hamsters were anesthetized using 5% isoflurane, and inoculated with 5 × 10⁵ PFU of SARS-CoV-2 (2019-nCoV/USA-WA1/2020) via the intranasal route, in a final volume of 100 μL. Baseline body weights were measured before infection as well as monitored daily for 7 and 11 days post infection in the prophylactic and therapeutic studies respectively. All experiments with the hamsters were performed in accordance with the NRC Guide for Care and Use of Laboratory Animals, the Animal Welfare act, and the CDC/NIH Biosafety and Microbiological and Biomedical Laboratories as well as the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia who also approved the animal experimental protocol. All animal studies infection with SARS-CoV-2 were conducted in the Animal Health Research Center (AHRC) Biosafety Level 3 (BSL-3) laboratories of the University of Georgia.

Determination of viral load by TCID₅₀ assay

Lung tissues were homogenized in 1 mL of DMEM containing 1% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The lung homogenate supernatant was diluted 10-fold (10° to 10⁶) and used to determine median tissue culture infection dose (TCID₅₀) in Vero E6 cells as previously described (Jang and Ross, 2020).

Human IgG detection in hamster sera

ELISA assay was used to detect the human IgG J08-MUT in hamster sera. 384-well plates (384 well plates, Microplate Clear; Greiner Bio-one) were coated with 2 μg/mL of unlabled goat anti-human IgG (SouthernBiotech) diluted in sterile PBS and incubated at 4°C overnight. 50 μL/well of saturation buffer (PBS/BSA 1%) was used to saturate unspecific binding and plates were incubated at 37°C for 1 h without CO₂. Hamster sera were diluted in PBS/BSA 1%/Tween20 0.05% at a starting dilution of 1:10. Fourteen reciprocal dilutions were performed. Alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich) was used as secondary antibody and pNPP (p-nitrophenyl phosphate) (Sigma-Aldrich) was used as soluble substrate. Wells were washed three times between each step with PBS/BSA 1%/Tween20 0.05%. The final reaction was measured by using the Varioskan Lux Reader (Thermo Fisher Scientific) at a wavelength of 405 nm. Samples were considered as positive if OD at 405 nm (OD₄₀₅) was twice the blank.