The aim of the study was to identify SARS-CoV-2 human monoclonal antibody with potent and broad neutralizing activity against SARS-CoV-2 and variants of concern. We immunized H2L2 transgenic mice carrying human variable heavy and light chain immunoglobulin genes with the SARS-CoV-2 spike ectodomain and receptor binding domain. We generated hybridoma’s from isolated B-cells, and used pseudovirus screening to assess neutralizing activity of supernatants from hybridoma’s against pseudoviruses carrying SARS-CoV-2 S protein with E484K substitution, a residue that is of variance in VOCs with immune escape potential. We used multiple methods including ELISA, biolayerinterferometry, single-particle cryo-EM reconstruction, site-directed mutagenesis and receptor-binding inhibition assays to characterize the antibody binding kinetics and affinity, epitope location and mechanism of neutralization. Pseudovirus and live virus assays were used to assess antibody-mediated neutralization of authentic SARS-CoV-2 and VOCs. We used mouse and hamster infection models to evaluate the antibody-mediated protective efficacy against challenge with SARS-CoV-2 and VOCs. At euthanasia infectious virus in lung and nasal turbinate tissues was quantified on cultured cells. The pathology in lung and nasal tissues was evaluated using histology and viral antigens by immunohistochemistry.
Viruses and cells. Calu-3 cells were maintained in Opti-MEM I (1) + GlutaMAX (Gibco) supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator. HEK-293T cells were cultured in DMEM supplemented with 10% FCS, sodium pyruvate (1 mM, Gibco), non-essential amino acids (1×, Lonza), penicillin (100 IU/mL), and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator. Cell lines tested negative for mycoplasma. SARS-CoV-2 isolates were grown to passage 3 on Calu-3 cells. For stock production, infections were performed at a multiplicity of infection (moi) of 0.01 and virus was collected at 72 hours post-infection, clarified by centrifugation and stored at -80°C in aliquots. All work with infectious SARS-CoV-2 was performed in a Class II Biosafety Cabinet under BSL-3 conditions at Erasmus Medical Center. Viral genome sequences were determined using Illumina deep-sequencing as described before (38). The 614G virus (clade B; isolate Bavpat-1; European Virus Archive Global #026 V-03883) passage 3 sequence was identical to the passage 1 (kindly provided by Dr. Christian Drosten). The Alpha (B.1.1.7; MW947280), Gamma (P.1; OM442897), Delta (B.1.617.2; OM287123), Omicron BA.1 (B.1.1.529.1; OM287553), Omicron BA.2 (B.1.1.529.2), Lambda (C.37) and Mu (B.1.621) variant passage 3 sequences were identical to the original respiratory specimens. For Omicron, the S1 region of spike was not covered well due to primer mismatches. Therefore, the S1 region of the original respiratory specimen and passage 3 virus were confirmed to be identical by Sanger sequencing. The Beta variant (B.1.351; OM286905) passage 3 sequence contained two mutations compared the original respiratory specimen: one synonymous mutations C13860T (Wuhan-Hu-1 position) in ORF1ab and a L71P change in the E gene (T26456C, Wuhan-Hu-1 position). No other minor variants >40% were detected. SARS-CoV-2 variants of concern/interest used contained the following spike changes relative to the Wuhan-Hu-1 strain: Alpha (B.1.1.7), Δ69-70, Δ144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H; Beta (B.1.351), L18F, D80A, D215G, Δ241-243, K417N, E484K, N501Y, D614G, A701V; Gamma (P.1), L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F; Delta (B.1.617.2), T19R, G142D, E156G, Δ157-158, L452R, T478K, D614G, P681R, D950N; Omicron BA.1 (B.1.1.529.1), A67V, Δ69-70, T95I, G142D, Δ143-145, N211I, Δ212, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F; Omicron BA.2 (B.1.1.529.2), T19I, L24S, ∆25/27, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; Lambda (C.37), G75V, T76I, R246N, Δ247-253, L452Q, F490S, D614G, T859N; Mu (B.1.621) T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, D950N.
Expression and purification of SARS-CoV-2 S proteins. Human codon-optimized gene was synthesized at Genscript encoding the 6P-stabilized SARS-CoV-2 S ectodomain expression construct (25)(S protein residues 1–1,213, Wuhan-Hu-1 strain: GenBank: QHD43416.1) with a C-terminal T4 foldon trimerization motif followed by an octa-histidine tag and a Twin-Strep-tag® (39). Constructs encoding S1 (residues 1–682), the N-terminal domain (NTD, residues 1–294) or receptor-binding domain (RBD, residues 329–538) of SARS-CoV-2 S (Wuhan-Hu-1 strain), C-terminally tagged with Strep-tag have been described before (40). Human codon-optimized genes were synthesized encoding S1 proteins of Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs described above, including a C-terminal Strep-tag. All proteins were expressed transiently in HEK-293T (ATCC® CRL-11268) cells from pCAGGS expression plasmids, and secreted proteins were purified from culture supernatants using streptactin beads (IBA) following the manufacturer’s protocol. Spike variants with single-site residue substitutions were generated using Q5® High-fidelity DNA polymerase (NEB)-based site-directed mutagenesis.
Immunization, hybridoma culturing and production of (recombinant) monoclonal antibodies. Harbour H2L2 mice (Harbour BioMed) were immunized using heterologous DNA/protein immunization protocol 16-512-22 under animal license (AVD101002016512) approved by CCD (Dutch Central Comity for animal experimentation). Mice were housed in SPF facility with cage enrichment, light switched on at 7:00 and switched off at 19:00 and with humidity at around 40%. Both female and male H2L2 mice were used. The female mice were housed up to 4 per individually ventilated cage (IVC), while males were in separate IVC cages to prevent fighting. Food was standard and water and food intake ad libitum. Mice were immunized intradermally three times bi-weekly with 50 micrograms of plasmid DNA encoding the Wuhan-Hu-1 SARS-CoV-2 S ectodomain trimer in 20 microliters of water, using the AgilePulse Intradermal electroporator system (BTX) according to the manufacturer instructions. After priming with DNA, immunization was continued in bi-weekly intervals by subcutaneous and/or intraperitoneal injection of 20-30 μg of antigen preparations formulated with Ribi Adjuvant System (RAS, Sigma) according to manufacturer instructions, alternating between the S ectodomain trimer and RBD of Wuhan-Hu-1 SARS-CoV-2 as antigens. Antigen specific antibody titers were monitored during immunization by taking blood samples from the mice and performing antigen-specific ELISA. High-titer mice were euthanized three to five days after the last protein boost (6 in total), and lymphoid organs (spleen and lymph nodes) were dissected. Single cell suspensions were generated by gently pressing and moving the lymphoid tissue in the presence of RPMI 1640 medium with a 2 ml syringe rubber part plunger against the 40 μm nylon cell strainer (Falcon) fitted in a 50 ml conical tube. Single cells were then fused to generate hybridomas by Polyethylene Glycol (PEG) mediated fusion with Sp 2/0 myeloma cells (ATCC #CRL-1581). In short, separate fusions were done for each mouse (spleen and lymph node cells), cells were washed in Fusion medium (RPMI 1640) and transferred to the new 50 ml tube. Sp 2/0 cells were cultured in advance (RPMI, 10% FCS, L glut, penicillin (100 IU/mL), and streptomycin (100 IU/mL), 0.4% Hybridoma Fusion and Cloning Supplement (HFCS)). For each fusion we used 1.0 × 108 Sp2/0 cells. Sp2/0 cells were washed in Fusion medium, and added to the tube with single cell suspension of splenocytes/ lymphocytes from one mouse (~2.0-2.5 × 108 cells). Fusion medium was added to the 50 ml mark. After centrifugation (5 min at at 400 g), supernatant was discarded and pellet was disrupted by gently tapping the bottom of the tube. Tube was placed at 37°C and 1 ml of pre-warmed PEG (PEG 1500, Roche) was slowly added over a period of 1 min to the cell pellet by dripping down the side of the tube under gentle stirring. After additional incubation for 1.5 min, 2 ml of pre-warmed Fusion medium was added to the tube over a period of 2 min under gentle stirring. An additional 14 ml of Fusion medium was added to the tube over a period of 2 min under gentle stirring, and the tube was left for 10 min at 37°C. Cells were spun down, re-suspended in 300 ml of selection medium (advanced RPMI 1640, GlutaMAX, 2% HFCS, 0.4μM aminopterin, 100μM Hypoxantine, 16μM Thymidine, 10% FCS, penicillin (100 IU/mL) and streptomycin (100 IU/mL)), and plated in 96 well plates (200 microliters per well). On day 6-7 we added 100 μl of post-fusion selection medium to each well. Screening of hybridoma supernatants was started 10-14 days post fusion. Supernatants from 96 well plates (estimated to have 1-4 hybridoma clones per well) were screened for SARS-CoV-2 S binding antibodies by ELISA using SARS-CoV-2 S ectodomain-coated plates (see below), and for neutralizing antibodies using the SARS-CoV-2 pseudovirus neutralization assay (see below). Selected hybridomas were subcloned by limited dilution and retested in ELISA and pseudovirus assay.
Production of recombinant human antibodies using HEK-293T was described previously (41). Briefly, gene blocks encoding the variable heavy (VH) and light (VL) chain sequences of 87G7 and of benchmark monoclonal antibodies REGN10933, REGN10987 (PDB ID: 6XDG) (42), S309 (PDB ID: 6WPS) (43), CR3022 (GenBank accession numbers: DQ168569.1 and DQ168570.1) (44), 47D11 (GenBank accession numbers: MW881223.1 and MW881224.1) (40) were synthesized. VH and VL sequences were separately cloned into the expression plasmids with human IgG1 heavy chain and human kappa chain constant regions, respectively using the HBM vectors pHBM 000254 (VH into pTT5-mIGK- hIgG1_HCv2) and HBM 000265 (VK into pTT5mIgK-hIgG_KCv2). Recombinant human antibodies were expressed in HEK-293T cells following transient transfection using polyethylenimine with pairs of the IgG1 heavy and light chain expression plasmids. At 18 hours after transfection, the transfection mixture was replaced by 293 SFM II expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter), penicillin (100 IU/mL), and streptomycin (100 IU/mL), GlutaMAX and 1.5% DMSO. Tissue culture supernatants were harvested 5–6 days after transfection and recombinant antibodies were purified using Protein A Sepharose (IBA) according to the manufacturer’s instructions.
ELISA analysis of antibody binding to SARS-CoV-2 S antigens. Purified S antigens (1μg/ml) were coated onto 96-well NUNC Maxisorp plates (Thermo Scientific) at room temperature (RT) for 3 hours followed by three washing steps with Phosphate Saline Buffer (PBS) containing 0.05% Tween-20. Plates were blocked with 3% bovine serum albumin (BSA, Fitzgerald) in PBS with 0.1% Tween-20 at 4°C overnight. Antibodies in hybridoma supernatants diluted in PBS containing 3% BSA and 0.1% Tween20 were allowed to bind to the ELISA plates at RT for 1 hour and binding was determined using a 1:3000 diluted HRP-conjugated mouse anti-rat IgG1, IgG2b and IgG2 mix (Absea) for 1 h at RT. Alternatively, 87G7 mAb was allowed to bind to the plates at 5-fold serial dilutions, starting at 10 μg/ml diluted in PBS containing 3% BSA and 0.1% Tween20, at RT for 1 hour. Antibody binding to the S proteins was determined using a 1:2000 diluted HRP conjugated goat anti-human IgG (ITK Southern Biotech) for 1 hour at RT. HRP activity was measured at 450 nm using tetramethylbenzidine substrate (BioFX) and an ELISA plate reader (EL-808, Biotek).
Antibody binding kinetics and affinity measurement. 87G7 (21 nM) was loaded onto Protein A biosensors (ForteBio) for 10 min. Antigen binding was performed by incubating the biosensor with 2-fold dilutions of recombinant SARS-CoV-2 S1 monomer or S ectodomain trimer for 10 min followed by a long dissociation step (30 min) to observe the decrease of the binding response. The affinity constant KD was calculated using 1:1 Langmuir binding model on Fortebio Data Analysis 7.0 software.
Biolayer interferometry-based binding competition assay. Binding competition was performed using biolayer interferometry (Octet Red348; ForteBio), as described previously (40, 41). In brief, SARS-CoV-2 S ectodomain trimer (50 μg/ml) was immobilized onto the anti-strep mAb-coated protein A biosensor. After a brief washing step, the biosensor tips were immersed into a well containing primary mAb (50 μg/ml) for 15 min and subsequently into a well for 15 min containing the competing mAb (secondary mAb; 50 μg/ml) or recombinant soluble ACE2. A 3 to 5-min washing step in PBS was included in between steps.
ELISA-based receptor-binding inhibition assay. The ACE2 receptor-binding inhibition assay was performed as described previously (40, 41). Recombinant soluble ACE2 was coated on NUNC Maxisorp plates (Thermo Scientific) at 1μg/well at RT for 3 h. Plates were washed three times with PBS containing 0.05% Tween-20 and blocked with 3% BSA (Fitzgerald) in PBS containing 0.1% Tween-20 at 4 °C overnight. Recombinant SARS-CoV-2 S RBD domain (200 nM) and serially diluted mAbs were mixed and incubated for 2 h at RT. The mixture was added to the plate for 2 h at 4 °C, after which plates were washed three times. Binding of SARS-CoV-2 S RBD domain to ACE2 was detected using 1:2000 diluted HRP-conjugated anti-StrepMAb (IBA) that recognizes the Strep-tag affinity tag on the SARS-CoV-2 S RBD domain. Detection of HRP activity was performed as described above (ELISA section).
Pseudovirus neutralization assay. Human codon-optimized genes encoding the spike proteins of SARS-CoV-2 S proteins corresponding to ancestral Wuhan-Hu-1 virus (GenBank: NC_045512.2) or variants of concern Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) were synthesized by GenScript. The production of SARS-CoV-2 S pseudotyped vesicular stomatitis virus (VSV) and the neutralization assay were performed as described previously (40). In brief, HEK-293T cells at 70~80% confluency were transfected with the pCAGGS expression vectors encoding SARS-CoV-2 S with a C-terminal cytoplasmic tail 18-residue truncation to increase cell surface expression levels. Cells were infected with VSV G pseudotyped VSVΔG bearing the firefly (Photinus pyralis) luciferase reporter gene at 48 hours after transfection. Twenty-four hours later, the supernatant was harvested and filtered through 0.45 μm membrane. Pseudotyped VSV was titrated on VeroE6 cells. In the virus neutralization assay, 3-fold serially diluted mAbs were pre-incubated with an equal volume of virus at RT for 1 hour, and then inoculated on VeroE6 cells, and further incubated at 37°C. Alternatively, pseudovirus was preincubated with 1/10 volume of H2L2 hybridoma culture supernatant for 1h, prior to infection of Vero cells. After 20 hours, cells were washed once with PBS and lysed with Passive lysis buffer (Promega). The expression of firefly luciferase was measured on a Berthold Centro LB 960 plate luminometer using D-luciferin as a substrate (Promega). The percentage of neutralization was calculated as the ratio of the reduction in luciferase readout in the presence of mAbs normalized to luciferase readout in the absence of mAb. The half maximal inhibitory concentrations (IC50) were determined using 4-parameter logistic regression (GraphPad Prism v8.3.0).
Live virus neutralization assay. Human monoclonal antibodies were tested for live virus neutralization using a plaque reduction neutralization (PRNT) assay. PRNT was performed according to a previously published protocol (38), with minor modifications. Briefly, 50 μl of serially diluted antibody in Opti-MEM I (IX) + GlutaMAX (Gibco, USA) was mixed 1:1 with virus (400 PFU) and incubated at 37°C for 1 hour before layering over fully confluent monolayers of Calu-3 cells (washed once prior with Opti-MEM I (IX) + GlutaMAX). After 8 hours of infection, the cells were fixed with formalin, permeabilized with 70% ethanol, washed in PBS and stained using rabbit anti-SARS-CoV nucleocapsid (SinoBiological, 1:2000 in 0.1% bovine serum albumin (BSA) in PBS) followed by goat anti-rabbit Alexa Fluor 488 antibody (Invitrogen, 1:2000 in 0.1% BSA in PBS). Plates were scanned on the Amersham Typhoon Biomolecular Imager (GE Healthcare, USA). Data were analyzed using ImageQuantTL 8.2 image analysis software (GE Healthcare). The PRNT titer was calculated using Graphpad Prism 9, calculating a 50% reduction in infected cells counts based on non-linear regression with bottom constraints of 0% and top constraints of 100%.
Cryo-electron microscopy sample preparation and data collection. The 87G7 Fab fragment was digested from the IgG with papain using a Pierce Fab Preparation Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Spike-Fab complexes were prepared under two conditions. For the first condition, 4 μl of SARS-CoV-2 hexaproline spike ectodomain, at a concentration of 28 μM (based on the molecular weight of the spike protomer) was combined with 1 μl of 150 μM 87G7 Fab and incubated for ~10 min at RT before blotting and plunge freezing. For the second condition, 3.5 μl of 28 μM SARS-CoV-2 hexaproline spike ectodomain was combined with 1 μl of 150 μM 87G7 Fab and then incubated for ~10 min at RT. Immediately before blotting and plunge freezing, 0.5 μl of 0.2% (w/v) fluorinated octyl maltoside (FOM) was added to the sample, resulting in a final FOM concentration of 0.02% (w/v). For both conditions, 3 μl of spike-Fab complex was applied to glow-discharged (20 mAmp, 30 s, Quorum GloQube) Quantifoil R1.2/1.3 grids (Quantifoil Micro Tools GmbH), blotted for 5 s using blot force 2 and plunge frozen into liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific). The data were collected on a Thermo Scientific Krios G4 Cryo Transmission Electron Microscope (Cryo-TEM) equipped with Selectris X Imaging Filter (Thermo Fisher Scientific) and Falcon 4 Direct Electron Detector (Thermo Fisher Scientific) operated in Electron-Event representation (EER) mode. Data processing was performed in Relion 3.1 (45) and cryoSPARC (46) single particle analysis suites. Raw data were imported in cryoSPARC. After Patch motion correction and Patch CTF estimation, 313,636 particles were picked from 1331 images from 0.02% FOM dataset and 621,175 particles were picked from 2500 images without FOM. After 2D classification and heterogeneous refinement, the best particle stack consisting of 133,550 particles was subjected to non-uniform refinement (47) with C3 symmetry imposed yielding a Spike-Fab complex cryo-EM map with an overall resolution of 2.9 Å. Following global refinement, a soft mask encompassing one RBD with the Fab bound was made in UCSF Chimera (48). Particles were imported into Relion 3.1 and, using the “relion_particle_symmetry_expand” tool, each particle from the C3-symmetry–imposed reconstruction was assigned three orientations corresponding to its symmetry related views. The soft mask was placed over a single RBD-Fab region of the map, and the symmetry- expanded particles were subjected to masked 3D classification without alignment using a regularization parameter (‘T’ number) of 20. Following a single round of focused classification, the best particle stack consisting of 72,118 particles was imported back to cryoSPARC and refined without imposing symmetry using the local refinement job, yielding a map with a global resolution of 4.9 Å. The nominal resolutions and local resolution estimations for the global and local refinements were performed in Relion 3.1. The ‘Gold Standard’ Fourier shell correlation (FSC) criterion (FSC = 0.143) was used for resolution estimates. Finally, the globally and locally refined maps were masked and sharpened using DeepEMhancer tool (49), as implemented in COSMIC2 (50), and combined using the “vop add” command in UCSF Chimera (48). Data collection and reconstruction parameters can be found in Table S1.
Model building and refinement. UCSF Chimera (48) (version 1.15.0) and Coot (51) (version 0.9.6) were used for model building. As a starting point for modelling the 87G7-bound spike, the crystal structure of the SARS-CoV-2-S N-terminal domain (residues 14-308; PDB ID: 7B62 (52)), the fully open SARS-CoV-2-S model (residues 309-332 and 527-1145; PDB ID: 7K4N (28)) and RBD crystal structure (residues 333-526; PDB ID 6M0J (53)) were individually rigid-body fitted into the composite density map using the UCSF Chimera “Fit in map” tool (48). Subsequently, the models were combined, and the peptide sequence was adjusted to match the 6P spike construct used in this study. For modelling the 87G7 Fab fragment, atomic coordinates of the heavy chain (HC) and the light chain (LC) variable regions were generated using the phyre2 server (54) and rigid body fitted into the EM density map using the UCSF Chimera ‘fit in map’ tool and then combined with the spike model. The resulting model was then edited in Coot using the ‘real-space refinement (51), carbohydrate module (55) and ‘sphere refinement’ tool. Iterative rounds of manual fitting in Coot and real space refinement in Phenix (56) were carried out to improve non-ideal rotamers, bond angles and Ramachandran outliers. During refinement with Phenix, secondary structure and non-crystallographic symmetry restraints were imposed. The final model was validated with MolProbity (57), EMRinger (58) and Privateer (glycans) (59, 60).
Analysis and visualization. Spike residues interacting with 87G7 were identified using PDBePISA (61) and LigPlot+ (62). Surface coloring of the SARS-CoV-2 RBD according to sequence conservation and the Kyte-Doolittle hydrophobicity scale was performed in UCSF ChimeraX (63). The UCSF Chimera “MatchMaker” tool was used to obtain RMSD values, using default settings. Figures were generated using UCSF Chimera (48) and UCSF ChimeraX (63). Structural biology applications used in this project were compiled and configured by SBGrid (64).
Mouse challenge experiment. In vivo prophylactic and therapeutic efficacy of mAb 87G7 against challenge with SARS-CoV-2 and four variants of concern, was evaluated in heterozygous K18-hACE2 C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) obtained from The Jackson Laboratory. Groups of 14-week-old female mice (n = 5), were given 200 μg of 87G7 or isotype control antibody (equivalent to 10 mg of the antibody per kg) by intraperitoneal injection, 16 hours before or one day after intranasal inoculation with a lethal dose of the indicated SARS-CoV-2 strain (105 PFU/mouse). Virus inoculations were performed under anesthesia that was induced with isoflurane, and all efforts were made to minimize animal suffering. All animals were housed in a self-contained ventilated rack (Tecniplast, IT), with the light switched on at 7:30 and switched off at 19:30. The ambient temperature was 19.5-22 °C and with humidity at 35-40%. Animal protection studies were carried out under the animal permit PROEX-146.6/20, approved by the Community of Madrid (Spain), and performed in biosafety level 3 facilities at CISA-INIA (Madrid).
To quantify infectious SARS-CoV-2 virus particles, one fourth of the right lung was homogenized using a MACS homogenizer (Miltenyi Biotec) according to the manufacturer’s protocols. Virus titrations were done using plaque assay performed on Vero E6 cells following standard procedures. In brief, cells were overlaid with DMEM containing 0.6% low-melting agarose and 2% FBS, fixed with 10% formaldehyde and stained with 0.1% crystal violet at 72 h post-infection.
To quantify viral antigen by immunohistochemistry, left lung lobes were fixed in 10% buffered formalin (Chemie Vertrieb GmbH & Co Hannover KG, Hannover, Germany). Left lung lobes were pre-fixed by injections of 10% buffered formalin as recommended by Meyerholz et al. (65) to ensure an optimal histopathological evaluation (Table S2).
Hamster challenge experiment. During the experiment, the animals were under veterinary observation and all efforts were made to minimize distress. Approval for the experiments was given by the German Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES file number 21/3755) and by the Dutch authorities (Project license number 27700202114492-WP12). Syrian hamsters (Mesocricetus auratus, 6-10 weeks old, Janvier Labs) were housed under BSL-3 conditions, starting 10 days prior to the experiment. 87G7 or a non-SARS-CoV-2 human IgG control antibody were injected intraperitoneally in a volume of 500 μl. The hamsters were challenged intranasally, 24 hours after or 12 hours before antibody inoculation, with 104 TCID50 of the respective SARS-CoV-2 variants, respectively. The animals were monitored for body weight loss and clinical symptoms twice daily until they were humanely euthanized four days after infection. Antibody injection, with challenge virus and euthanasia were performed under isoflurane anesthesia. Left nasal turbinates and left lung lobe were fixed in 10% buffered formalin (Chemie Vertrieb GmbH & Co Hannover KG, Hannover, Germany) from the investigated hamsters. Left lung lobes were pre-fixed by injections of 10% buffered formalin (65) to ensure an optimal histopathological evaluation. Left nasal turbinates, following formalin fixation, were decalcified in soft tissue decalcifier (Roth # 6484.2) for about 14 days prior to routine tissue processing.
To quantify infectious SARS-CoV-2 virus particles, lung and nasal turbinate tissues were homogenized using a TissueLyser II (Qiagen) and infectious SARS-CoV-2 virus particles in tissue homogenates were quantified on Vero E6 cells. Cells were infected with 10 fold serial dilutions of the homogenized tissue prepared in DMEM + 2% FBS (starting dilution 100- and 10-fold for lung and nasal turbinate homogenate, respectively). Plates were further incubated in a humidified atmosphere, at 37°C, 5% CO2. Cytopathic effect was evaluated 5 days post infection. Omicron samples were titrated in Calu-3 cells due to the low infectivity of Omicron in Vero cells. In this case, after 5 day incubation, cells were fixed with 4% PFA and stained using an anti-SARS-CoV-2 nucleocapsid antibody (Sinobiological). Virus titers (TCID50/ml) were calculated using the Spearman-Karber method.
Formalin-fixed, paraffin-embedded (FFPE) tissue was used for histology and immunohistochemistry. Histopathological lesions were evaluated on hematoxylin-eosin (HE) stained sections. For the detection of viral antigen in Syrian golden hamsters, immunohistochemistry with a monoclonal antibody detecting SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological 40143-MM05) was performed on FFPE tissue sections, as described previously (66, 67). Briefly, tissue sections were dewaxed and rehydrated, followed by endogenous peroxidase blocking for 30 min at RT. Antigen retrieval was performed in Na2H2EDTA buffer for 20 min in a microwave at 800 W. The primary antibody (dilution 1:4000) was applied for 1 hour at RT. Sections were subsequently rinsed, and secondary labeling was performed using the respective peroxidase-labeled polymer (Dako Agilent Pathology Solutions, K4003) for 30 min for 60 min at RT. Visualization of the reaction was accomplished by incubation in chromogen 3,3-diaminobenzidine tetrahydrochloride (DAB, 0.05%) and 0.03% H2O2 in PBS for 5 min. The slides were afterwards counterstained with Mayer’s hematoxylin for 1 min. Nasal turbinates were evaluated on a full-length longitudinal section of the nose including respiratory and olfactory epithelium. Assessment of histopathological lesions in the nasal turbinates was performed with a semiquantitative score system, as described previously with minor modifications. Quantification of the viral antigen in the nasal epithelium was performed using a semiquantitative score. Hamsters left lung lobe was evaluated on one cross-section (at the level of the entry of the main bronchus) and one longitudinal section (along the main bronchus) of the entire left lung lobe. Assessment of histopathological lesions and viral load in the lung was performed with a semiquantitative scoring system, as described previously with minor modifications (68). System for semiquantitative scoring of histopathological lesions and viral antigen in nose and lung is shown in Table S3-S5. Histopathological semiquantitative evaluations were performed by veterinary pathologists (GB, MC, FA) and subsequently confirmed by a European board certified veterinary pathologist (WB). During the evaluation, the pathologist was blinded regarding the treatment groups and used virus strains.
Du W., Hurdiss D.L., Drabek D., Mykytyn A.Z., Kaiser F.K., González-Hernández M., Muñoz-Santos D., Lamers M.M., van Haperen R., Li W., Drulyte I., Wang C., Sola I., Armando F., Beythien G., Ciurkiewicz M., Baumgärtner W., Guilfoyle K., Smits T., van der Lee J., van Kuppeveld F.J., van Amerongen G., Haagmans B.L., Enjuanes L., Osterhaus A.D., Grosveld F, & Bosch B.J. (2022). An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern. Science Immunology.
