Production of recombinant RBD
SARS-CoV-2 RBD was produced in HEK293T cells from a clone kindly provided by Prof. Florian Kramer (Icahn School of Medicine at Mt. Sinai) and purified as reported58 with a slight modification to purify the monomeric form of RBD used in this study. The protein was further purified on a Superdex 75 column (Cytiva) pre-equilibrated with phosphate buffered saline.
In vivo vaccination study
Animals were cared for following federal, state, and local guidelines. The University of Michigan, Ann Arbor is an AAALAC international accredited institution, and all works conducted on animals were in accordance with and approved by the Institutional Animal Care and Use Committee (IACUC). All animal experiments were carried out in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Female BALB/c (n = 5 per group) mice 5–6 weeks of age were purchased from Jackson Laboratory. Mice were used without further randomization. Mice (n = 5 per group) were given a week of acclimation and were vaccinated 3 times with a 2-week interval. Each dose containing 0.5 µg of RBD and 500 µg of alum (Alhydrogel, Invivogen) was subcutaneously injected at the tail base. Immune sera were collected on weeks 4, 6, and 10 and analyzed for RBD-specific IgG and IgG1 antibody titers by ELISA. Briefly, RBD protein was coated on 96-well ELISA plates (0.1 µg/well), and serially diluted sera samples were added. After an hour of incubation and multiple washings, horseradish peroxidase (HRP)-labeled secondary antibodies were added and incubated for 1 h at room temperature. Secondary antibodies used were rabbit anti-mouse IgG H&L-HRP (Abcam) and goat anti-mouse IgG1-HRP (Southern Biotech). TMB substrate solution was added, and the reaction was stopped by the addition of NaF. The absorbance was measured at a 620 nm wavelength using a plate reader (Synergy Neo, BioTek). To measure antibody titers, titration curves were evaluated based on the absorbance and the dilution factor, from which half maximal effective concentration (EC50) values were calculated using software Gen5 (BioTek). A subset of mice was used for spleen harvest on week 6.
B cell isolation and staining
To isolate B cells, freshly excised mouse spleens were washed with ice-cold FACS buffer (HBSS no calcium or magnesium with 1 mM EDTA, 25 mM HEPES, 1% FBS) and processed through a 70 µm cell strainer. Cells were washed once with ice-cold FACS buffer, and then treated with AKC lysis buffer for 2 min to lyse red blood cells. Cells were then washed again with FACS buffer and passed through a 40 µm cell strainer. Magnetic-activated cell sorting (MACS) was then performed to enrich for B cells based on CD45R expression. Briefly, splenocytes were incubated with mouse FcR blocking reagent (Miltenyi Biotec, 130-092-575) following the manufacturer’s protocol. Splenocytes were then incubated with mouse CD45R (B220) microbeads (Miltenyi Biotec, 130-049-501) and MACS was performed following the manufacturer’s protocol using LS columns (Miltenyi Biotec, 130-042-401) and a Midi MACS separator (Miltenyi Biotec, 130-042-302).
Enriched cells were counted and prepared for fluorescence-activated cell sorting (FACS) by adding a mixture of fluorescently labeled antibodies and antigen. Labeling antibodies were added at 1:1000 dilution in a volume of 1 mL per 107 cells for 30 min at room temperature. The following labeling scheme was employed, listed in the format of ‘molecular target (fluorophore)’: CD19 (AF700), IgG1 (BV421), CD4 (FITC), CD8 (FITC), GR-1 (FITC), F4/80 (FITC), IgM (PE Cy7). SARS-CoV-2 RBD-PE and RBD-APC were each added at 1 µg per 108 cells. Prior to the assay, SARS-CoV-2 RBD (Acro Biosystems, SPD-C52H3) was labeled using Lightning Link PE and APC labeling kits (Novus Biologics, 705–0030 and 703–0030) following the manufacturer’s protocol. Cells (5 × 105) were reserved for each single color compensation control, and labeling antibodies and RBD antigen were likewise applied at 1:1000 dilution for 30 min at room temperature. Following the antibody labeling/antigen incubation step, cells were washed twice with cold FACS buffer. Dead cell marker 7-Aminoactinomycin D (7-AAD) (Invitrogen, number A1310) was added 10 min prior to cell sorting.
The following labeling antibodies were used for FACS preparation: rat anti-mouse IgG brilliant violet 421 (Clone A85-1; BD biosciences, 562580), rat anti-mouse IgM PE-Cy7 (PE/Cy7 anti-mouse IgM Antibody Clone RMM1; Bio legend, 406513), rat anti-mouse IgD APC-Cy7 (APC/Cy7 anti-mouse IgD Antibody, Clone 11-26c; BioLegend, 405715), rat anti-mouse CD19 AF700 (Alexa Fluor® 700 Rat anti-Mouse CD19, Clone 1D3; BD biosciences, 557958), rat anti mouse CD4 FITC (FITC Rat Anti-Mouse CD4, Clone GK1.5; BD Biosciences, 557307), rat anti mouse CD8 FITC (FITC Mouse Anti-Rat CD8a, Clone OX-8; BD Biosciences, 561965), rat anti mouse GR1 FITC (FITC Rat Anti-Mouse Ly-6G and LY-6C, Clone RB6-8C5; BD Biosciences, 553126), rat anti mouse F4/80 FITC (F4/80 Monoclonal Antibody BM8, FITC, eBioscience; Thermo Fisher, 11-4801-82).
The following labeling antibodies were used for single color compensation controls: rat anti mouse CD45—Brilliant violet 421(Clone 30-F11; BD biosciences, 563890), rat anti mouse CD45—PE Cy7 (Clone 30-F11; BD biosciences, 561868), rat anti mouse CD45—APC Cy7 (Clone 30-F11; BD biosciences, 561037), rat anti mouse CD45—AF700 (Clone 30-F11; BD biosciences, 560510), rat anti mouse CD45—FITC (Clone 30-F11; BD biosciences, 553080), rat anti mouse CD45—PE (Clone 30-F11; BD biosciences, 553081), rat anti mouse CD45-APC (Clone 30-F11; BD biosciences, 553092).
Mouse single B cell sorting
Antigen-specific IgG1+ memory B cells were sorted single cell per well into skirted 96-well plates containing 4 µl of cell lysis solution20 using a BD Moflo Astrios Cell Sorter. The following gating strategy was applied in series: selection of lymphocytes based on size (forward scatter area) and granularity (side scatter area), doublet discrimination based on forward scatter width and forward scatter area, viability and lack of non-B cell markers (T cell marker CD4, T cell marker CD8, neutrophil marker GR-1, macrophage marker F4/80), B cell marker CD19, negative for IgM (naïve B cell marker) and positive for IgG1, negative for IgD (naïve B cell marker), and binding to SARS-CoV-2 RBD labeled with PE and APC fluorescent proteins.
cDNA generation and PCRs
After sorting single B cells into a 96-well plate containing 4 μL of lysis buffer in each well, reverse transcription was performed by adding 7 μL of RT mix I (Table S2) to each well and incubating on a preheated thermocycler (65 °C) for 5 min20. The plates were then placed on ice for 5 min and 7 μL of RT mix II (Table S2) was added to each well. The plates were incubated on a thermocycler and were run under the following program to generate cDNA (Table S3). To dilute cDNA, 10 μL of nuclease-free water was added to each well. First, PCR amplification was performed for variable heavy and light chains in separate PCR reactions by mixing 38 μL of PCR mix I (Table S4) with 4 μL of cDNA. Amplification was performed on a thermocycler following the first PCR program (Table S4). A second PCR amplification step was performed by mixing 38 μL of seq-PCR mix (Table S5) with 4 μL of PCR product from the first amplification on a thermocycler following the seq-PCR program (Table S5). Seq-PCR product from each well was loaded onto an agarose gel (2% w/v) and run at 120 V for 30 min. The expected bands (450–500 bp) were purified, and PCR products were sent for Sanger sequencing using appropriate reverse primers for heavy and light chains.
Cloning and recombinant antibody expression
Antibody VH genes were digested with EcoRI-HF (New England Biolabs, R3101L) and NheI-HF (New England Biolabs, R3131L), whereas VL genes were digested with EcoRI-HF and BsiWI-HF (New England Biolabs, R3553L) and purified (Qiagen, 28104). Expression plasmids (VH or VL) were digested with appropriate restrictions enzymes, following the manufacturer’s protocol, and subsequently treated with calf intestinal alkaline phosphatase (New England Biolabs, M0525L). The digested vector was analyzed by electrophoresis using a 1% agarose gel. Appropriate sized DNA was excised and purified. Digested inserts and vectors were ligated with T4 ligase (New England Biolabs, M0202L) and transformed via heat shock into DH5α chemically competent cells. Cells were incubated in the presence of LB media (antibiotic free) for 1 h at 37 °C (shaking at 200 rpm), and then plated on LB plates supplemented with ampicillin (100 µg/mL) overnight at 37 °C. Single colonies were picked, grown in LB media with ampicillin overnight, miniprepped (Qiagen, 27106), and sequenced via Sanger sequencing. The plasmids for the dual variable domain tetravalent antibody were similarly cloned, expressed, and purified.
The antibodies used in this study were expressed in HEK293-6E cells from National Research Council of Canada. The antibody heavy and light chain plasmids (7.5 μg each) were mixed with PEI (45 μg) at room temperature with F17 media (without supplements) for 10–15 min and added to cells at a density of 1.5–2 million cells per mL. Cell media was enhanced with 20% w/v yeastolate (BD Sciences, 292804) 24–48 h post transfection. Cells were grown for an additional 5 days at 37 °C in F17 media containing supplements: Glutamine (Invitrogen, 25030081), Kolliphor (Thermo Fisher Scientific, NC0917244) and G418 (Thermo Fisher Scientific, 10131035). Cell suspensions were centrifuged at 3500×g for 40 min. Cell supernatant was transferred to new tubes, and 0.5–1 mL dry volume of Protein A beads (Thermo Fisher Scientific, 20333) per culture was added, followed by overnight gentle rocking at 4 °C. Protein A beads were separated from media using vacuum filter columns (Thermo Fisher Scientific, 89898). The beads were then washed with 75–150 mL of PBS. Antibodies were eluted from Protein A beads using 0.1 M glycine buffer (pH 3.0) and then neutralized with 1 M Tris to a final pH of 7.4. Antibodies were then filtered using 0.2 µm filters, aliquoted and stored at −80 °C. Antibody absorbance at 280 nm was measured and antibody size was evaluated by SDS-PAGE (Thermo Fisher Scientific, WG1203BOX). SDS-PAGE gel images were acquired using a lightbox (Kaiser Slimlite Plano 5000 K 8 × 11”) and camera (iPhone 11).
Affinity analysis
For affinity analysis, 0.3 µg of biotinylated RBD was immobilized on 3 × 107 streptavidin Dynabeads in PBSB (PBS with 1 g/L BSA) in a final volume of 1.2 mL. Protein and beads were incubated at room temperature for 2–3 days and then stored at 4 °C. For the binding study, beads were washed twice with PBSB and blocked with 10% milk in PBSB by end-over-end mixing at room temperature for 1 h followed by another wash with PBSB. 105 beads/well were incubated with varying concentrations of antibodies in 1% milk in PBSB at room temperature for 2–3 h. Post incubation, the beads were washed once by centrifugation, followed by incubation with goat anti-human IgG AF647 (Jackson ImmunoResearch, 109-605-098) on ice for 4 min. After labeling, beads were washed once with ice-cold PBSB and evaluated by flow cytometry.
SARS-CoV-2 pseudovirus neutralization assay
The pseudovirus preparation and SARS-CoV-2 neutralization assay were modified from a previous protocol22. To prepare virus particles, Lenti-X 293T cells (Takara, 632180) were seeded at 6 × 105 per well in 6-well plates in RPMI media containing supplements of 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were cultured at 37 °C with 5% CO2 until reaching a target confluency of 50–70%. Cells were then transfected using lipofectamine 2000 and third generation lentivirus plasmids: HDM-Hgpm2 plasmid (BEI number NR-52517) encoding HIV Gag-Pol under CMV promoter (0.22 µg), HDM-tat1b plasmid (BEI, NR-52518) encoding HIV Tat under CMV promoter (0.22 µg), pRC-CMV-Rev1b plasmid (BEI number NR-52519) encoding HIV Rev (0.22 µg), pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI number NR-52516) lentiviral transfer plasmid encoding co-expression of luciferase and ZsGreen (1.00 µg), pCMV3 SARS-CoV2 S Untagged Delta 19AA C-term plasmid encoding the SARS-CoV-2 spike (S) protein with a 19-amino acid deletion at the C-terminus (0.34 µg). Cell media was exchanged to fresh RPMI with 10% FBS and 1% P/S at 24 h post-transfection. Then, to isolate and concentrate SARS-CoV-2 pseudovirus (without ultracentriguation), cell supernatant was collected and pressed through a 0.45 µm filter at 72 h post-transfection. Lenti-X Concentrator (Takara, 631232) was added to supernatant at a volume ratio of 1:3 and incubated at 4 °C overnight. To concentrate pseudovirus, the mixture was centrifuged at 1500×g for 45 min. Supernatant was removed, and the virus pellet was resuspended in a volume of 50 µL of Opti-MEM per well of virus harvest.
To determine the quantity of Tissue Culture Infectious Units (TCIU) per mL of virus, 293T-ACE2 cells (BEI, NR-52511) were seeded at 10,000 cells per well in a 96-well plate in DMEM with 10% FBS and 1% P/S, at 37 °C and 5% CO2. 24 h after seeding, the cells were infected with various dilutions of virus, diluted in DMEM media in the presence of 5 µg/mL polybrene, 10% FBS, and 1% P/S. Representative cell counts per well were also determined at 24 h post seeding. Then, the percentage of ZsGreen-expressing cells was determined via flow cytometry using a Bio-Rad ZE5 cell analyzer and was further verified using fluorescence microscopy at 48 h post-infection.
For the pseudovirus neutralization assays, 293T-ACE2 cells were seeded at 10,000 cells per well in white bottom 96-well plates (Corning, 3917) in DMEM (10% FBS and 1% P/S) and cultured at 37 °C and 5% CO2. At 24 h post-seeding, 293T-ACE2 cells were treated with a final concentration of 5 µg/mL polybrene, and mixtures containing 350 TCIU SARS-CoV-2 pseudovirus per well and antibody treatments (fourfold serial dilutions). The mixtures of antibody and SARS-CoV-2 pseudovirus were incubated together for 1 h at 37 °C prior to addition to 293T-ACE2 cells. Next, neutralizing activity was determined via bioluminescence detection using a microplate reader at 48 h post-infection. For this procedure, luciferase substrate (Promega ONE-Glo, E6110) was used following the manufacturer’s protocol. Specifically, 96-well plates were equilibrated to room temperature for 10 min and the media volume in each well was reduced to 100 µL. Luciferase substrate was prepared and added (100 µL per well). The plates were incubated at room temperature for 10 min, and bioluminescence was measured (500 ms integration/well) using a Molecular Devices SpectraMax microplate reader.
Specificity analysis
To evaluate the affinity of antibodies, His-tag labeled RBD of SARS-CoV and SARS-CoV-2 virus were separately immobilized on microbeads (Thermo Fisher, 10103D). 96-well plates containing RBD-coated beads were incubated with either 13I1 antibodies or VHH-72 nanobodies over a range of concentrations (3 pM to 12.5 nM) in PBSB for 2 h at room temperature. After primary incubation, the beads were washed with ice-cold PBSB and incubated with goat anti-human Fc AF 488 (Jackson ImmunoResearch, 109-545-008) for 4 min on ice. Following secondary incubation, the beads were washed twice with ice-cold PBSB and analyzed by flow cytometry. Similarly, 13I1 coated beads were assessed for binding to WT SARS-CoV-2 S1 protein as well as the S1 protein of variants of concern (B.1.1.7 and B.1.351) using commercially-available antigens (Acro Biosystems; SPD-C52H3, SPD-C52Hn, SPD-C52Hp). Briefly, 13I1-coated microbeads were blocked in PBSB with 10% milk for 1 h, washed with PBSB, and incubated with SARS-CoV-2 S1 proteins in 96-well plate format for 3 h at 25 °C and 225 rpm. Plates were washed with PBSB, incubated with His-tag antibody (Invitrogen, PA-9531) for 30 min on ice, washed with PBSB, incubated with detection antibody (Jackson ImmunoResearch, 703-606-155) for 4 min on ice, washed with PBSB, and analyzed by flow cytometry.
Competitive binding analysis
To evaluate the epitope of 13I1, competitive binding analysis was performed with ACE2 receptor and other published SARS-CoV-2 antibodies. Biotinylated RBD (5 nM) was first incubated with soluble antibodies or ACE2 over a range of concentrations (0.05, 0.5, 5, 50 and 500 nM) at room temperature for 2 h. Next, the antibody-RBD complexes were incubated with 13I1-coated microbeads in PBSB with 1% milk at room temperature for 3 h. After incubation, beads were washed with cold PBSB followed by incubation with streptavidin AF647 (1:1000) for 4 min on ice. After secondary incubation, the beads were washed twice with cold PBSB and analyzed by flow cytometry.
Melting temperature analysis
Antibody melting temperatures were measured using differential scanning fluorimetry. Briefly, antibodies were prepared at 0.12 mg/mL in PBS and combined with Protein Thermal Shift Dye (Applied Biosystems, 4461146) at a volume ratio of 7:1 antibody:dye. Background samples were prepared by mixing 1 × PBS with dye at the same ratio. The average of 2–3 PBS-dye mixtures was used to calculate background signal. The antibody-dye and PBS-dye mixtures were added to clear 384-well plates. Plates were submitted to the University of Michigan Advanced Genomics core for analysis. The 384-well plate was centrifuged at 1000–2000 rpm for 1 min and inserted into an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Thermal cycle conditions analyzed increasing temperatures between 25 and 98 °C over 45 min. Background signals were subtracted from sample signals during analysis. Melting temperatures were determined from the temperatures at which the maximum signals were observed (first derivatives equal to zero). In the case of the DVD construct, the first local maximum value was used to determine the melting temperature when two transitions were observed.
Analytical size-exclusion chromatography
Antibody purity after Protein A purification was analyzed using size-exclusion chromatography with a Shimadzu Prominence HPLC System outfitted with a LC-20AT pump, SIL-20AC autosampler and FRC-10A fraction collector. Antibodies in 20 mM acetate (pH 5) were buffer exchanged into PBS (pH 7.4). For analytical SEC, 100 µL of sample (diluted to 0.1 mg/mL) was loaded onto the column (Superdex 200 Increase 10/300 GL column; GE, 28990944) and evaluated at a flow rate of 0.75 mL/min using a PBS running buffer supplemented with 200 mM arginine (pH 7.4). Absorbance at 280 nm signal was monitored and used for analysis. The percentage of protein monomer was evaluated by analyzing the area under the peak between the exclusion volume and solvent elution times (8 to 22 min).
Polyspecificity analysis
Polyspecificity reagent (PSR) was prepared as previously reported27. Briefly, CHO cells (109, Gibco, A29133) were pelleted, washed with PBSB, washed again with Buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 10% Glycerol, pH 7.2), and then pelleted. The cell pellets were resuspended in 5 mL of Buffer B with supplementary protease inhibitor (Sigma Aldrich, 4693159001). The cells were homogenized for 90 s (three 30 s cycles) and then sonicated for 90 s (three 30 s cycles). The cell suspension was then centrifuged at 40,000×g for 1 h. The supernatant was then removed and discarded.
The pellet (enriched cell membrane fraction) was suspended in Buffer B with a Dounce homogenizer for 30 strokes. Protein concentration was determined using a detergent compatible protein assay kit (BioRad, 5000116). The enriched membrane fraction was diluted to a concentration of 1 mg/mL in solubilization buffer (pH 7.2) containing 50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 1% n-dodecyl-b-d-maltopyranoside (Sigma Aldrich, D4641), and a protease inhibitor (Sigma Aldrich, 11873580001). The solution was then mixed overnight at 4 °C via end-over-end mixing. The soluble membrane protein fraction was then centrifuged at 40,000 xg for 1 h and the supernatant was collected. The final concentration of supernatant was measured again and diluted to 1.0 mg/mL.
Sulfo-NHS-LC-biotin (Thermo Fisher Scientific, PI21335) was dissolved in distilled water at ~ 11.5 mg/mL. Stock solutions of Sulfo-NHS-LC-biotin (150 mL) and PSR reagent (4.5 mL at 1.0 mg/mL) were mixed end-over-end at room temperature for 45 min. To quench the reaction, 10 mL of 1.5 M hydroxylamine at pH 7.2 was added. Biotinylated PSR was then aliquoted and stored at −80 °C.
Protein A-coated magnetic beads (Invitrogen, 88846) were washed three times with PBSB and then incubated with antibodies or nanobodies at various concentrations ranging from 0.03 × to 10 × of the saturated bead binding capacity for IgGs in 96-well plates (VWR, 650261) overnight at 4 °C. Antibody and nanobody concentrations were normalized by molarity to maintain the same Fc concentration across the samples. The IgG-coated beads were washed twice with PBSB, with centriguation at 2500×g for 4 min between washing steps. Next, the beads were suspended with a 10 × diluted solution of biotinylated PSR and incubated for 20 min on ice. Following this incubation, beads were washed once with PBSB and then incubated with a 1000 × dilution of streptavidin AF-647 (Invitrogen, S32357) and a 1000 × dilution of goat anti-human Fc[F(ab’)2] AF-488 (Invitrogen, H10120) for 4 min on ice. Beads were washed once, resuspended in PBSB, and evaluated via flow cytometry. The high and low non-specific binding control antibodies used in this assay have the variable regions of emibetuzumab and elotuzumab grafted onto a common IgG1 framework, respectively. The control antibodies were two-step purified by Protein A and SEC. Results from all replicates were normalized between 0 and 1 based on control antibodies.
