Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice

Significance Neutralizing antibodies are important for immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and as therapeutics for the prevention and treatment of COVID-19. We identified high-affinity nanobodies against SARS-CoV-2 receptor-binding domain and found that nanobody cocktails consisting of two noncompeting nanobodies were able to block ACE2 engagement with RBD variants present in human populations and potently neutralize both wild-type SARS-CoV-2 and the N501Y D614G variant at low concentrations. Prophylactic administration of nanobody cocktails reduced viral loads in mice infected with the N501Y D614G SARS-CoV-2 virus, showing that nanobody cocktails are useful as prophylactic agents against SARS-CoV-2.

4 Antibody concentration was determined by absorbance measurement at 280 nm using a Nanodrop and purity was determined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Microneutralization assay
SARS-CoV-2 isolate hCoV/Australia/VIC01/2020 was passaged in Vero cells and stored at −80 °C. The ability of nanobodies to neutralize the infectivity of 100 median tissue culture infectious doses (TCID50) of virus was assessed in a microneutralization assay as previously described (4) . Serial two-fold dilutions of nanobodies starting at 1:20 were incubated with SARS-CoV-2 in minimum essential media (MEM)/0.5% bovine serum albumin (BSA) at room temperature for 1 h. Residual virus infectivity was assessed in quadruplicate wells of Vero cells and viral cytopathic effect was read on day five. The neutralizing antibody titer was calculated using the Reed-Muench method as previously described (4).
RBD variants were expressed in Expi293 HEK cells (ThermoFisher), which were maintained in suspension at 37 °C and 8% CO2. Cells were transfected at a density of 3 x 10 6 with 1 µg of plasmid DNA for 1 mL of culture and ExpiFectamine TM 293 reagent diluted in Opti-Mem TM (ThermoFisher) following the manufacturer's protocol. 22 hours after transfection, ExpiFectamine TM 293 transfection Enhancer 1 and 2 (ThermoFisher) was added to transfected cells along with lupin peptone. Five days after transfection, the supernatant was collected by centrifugation, 10 mM MgCl2 was added to improve binding to the column and filtered through a 0.22 µm filter. RBD variants were purified by loading the supernatant onto a 1 mL Ni Excel column (GE Healthcare). Columns were equilibrated 5 and washed using DPBS. RBD variants were eluted using 300 mM imidazole, 100 mM NaCl DPBS buffer. A second purification step was performed by loading the eluate on a Superdex75 increase 10/300 GL gel filtration column (GE Healthcare), which was equilibrated with DPBS. Recombinant proteins were concentrated using Amicon Ultra-4, 10 kDa (Millipore). Protein concentration was determined by absorbance measurement at 280 nm using a Nanodrop and purity was determined using SDS-PAGE.
RBD variant multiplex bead cocktails was generated as previously described (5).
Briefly, each respective RBD protein was coupled to a different magnetic carboxylated bead region (Bio Rad) using a two-step carbodiimide reaction, at a ratio of 1 million beads to 5 µg of each RBD antigen. Plates were incubated for two hours on a plate shaker at RT, washed twice in 0.05% PBS Tween 20, and relative nanobody binding was detected using anti-human IgG R-Phycoerythrin (PE) Conjugate (#9040-09, Southern Biotech) at 1.3 µg/ml for two hours RT with shaking. Plates were then washed three times, sheath buffer was added to each well and plates were acquired on a FlexMap3D™ (Luminex Corporation). The binding of IgG-PE to each bead was detected as MFI (Median Fluorescence Intensity). RBD ACE2 inhibition assays were conducted using RBD variant multiplex bead cocktails (1000 beads of each bead region per well) in black, clear bottom 384-well plates. 20 µl of 25 µg/ml of Avi-Tagged biotinylated ACE2 along with 10 µl of nanobodies (final concentration of 80 nM per well, with subsequent 8-fold 1:4 titrations). Human ACE2 (residues 19-613) ectodomain with a C-terminal His-tag was expressed in Expi293 cells and purified using Ni-NTA and size-exclusion chromatography before being biotinylated using Bir-A (Avidity). Total final volume per well was 50 µl. Plates were then incubated for 2 hours on a plate shaker at RT then were washed twice in 0.05% PBS Tween 20. 40 µl of Streptavidin, R-Phycoerythrin Conjugate (SAPE) (#S866, Thermo Fisher) at 4 µg/ml was then added for 1 hour, followed by the addition of 10 µl of 10 µg/ml of R-Phycoerythrin, Biotin-XX Conjugate (#P811, Thermo Fisher), with incubation for an additional hour. Plates were then washed three times, sheath buffer was added to each well, 6 with plates left to shake for 10 minutes on a plate shaker prior to acquisition on a FlexMap3D™ (Luminex Corporation). The binding of ACE2, detected as phycoerythrinlabelled reporter is measured as MFI (Median Fluorescence Intensity).

Measurement of viral burden for in vivo studies
Three days post-infection, animals were humanely killed and lungs removed and homogenised in a Bullet Blender (Next Advance Inc) in 1 mL DME media (ThermoFisher) containing steel homogenisation beads (Next Advance Inc). Samples were clarified by centrifugation at 10,000 x g for 5 minutes before virus quantification by TCID50 and RT-qPCR assays. SARS-CoV-2 live virus quantification by TCID50 assay: SARS-CoV-2 lung TCID50 was determined by plating 1:7 serially-diluted lung tissue homogenate onto confluent layers of Vero cells (clone CCL81) in DME media (ThermoFisher) containing 0.5 µg/ml trypsin-TPCK (ThermoFisher) in replicates of six on 96-well plates. Plates were incubated at 37 °C supplied with 5% CO2 for four days before measuring cytopathic effect under light microscope. The TCID50 calculation was performed using the Spearman and

Mass spectrometry sample preparation and analyses
Tryptic digestion of gel separated Nanobody proteins.
Nanobody bands were excised and processed. Briefly gel bands were first destained in a solution of 100 mM NH4HCO3 / 50% ethanol for 15 minutes at room temperature with shaking at 750 rpm. Destaining was repeated twice to ensure removal of excess Coomassie.
Destained bands were dehydrated with 100% ethanol for 5 minutes and then rehydrated in 50 mM NH4HCO3 containing 10 mM DTT. Protein bands were reduced for 60 minutes at 56 °C with shaking then washed twice in 100% ethanol for 10 minutes to remove DTT.
Reduced ethanol washed samples were sequentially alkylated with 55 mM iodoacetamide in 50 mM NH4HCO3 in the dark for 45 minutes at room temperature. Alkylated samples were then washed with milli-Q water followed by 100% ethanol twice for 5 minutes to remove residual Iodoacetamide then vacuum-dried for 10 minutes. Alkylated samples were then rehydrated with 20 ng/µl trypsin (Promega) in 50 mM NH4HCO3 at 4 °C for 1 hour.
Excess trypsin was removed, gel pieces were covered in 40 mM NH4HCO3 and incubated overnight at 37 °C. Peptides were concentrated and desalted using C18 stage tips before analysis by LC-MS.

Identification of Nanobody derived peptides using reversed phase LC-MS.
Purified peptides were re-suspended in 0.1% TFA, 2% acetonitrile and separated using a two-column chromatography set up composed of a PepMap100 C18 20 mm x 75 μm trap and a PepMap C18 500 mm x 75 μm analytical column (Thermo Fisher Scientific).
Samples were concentrated onto the trap column at 5 μL/min for 5 minutes with Buffer A (0.1% formic acid, 2% DMSO) and then infused into a Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific) equipped with a FAIMS Pro interface at 300 nl/minute via the analytical column using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific). 125-minute gradients were run for each sample altering the buffer composition from 3% buffer B (0.1% formic acid, 77.9% acetonitrile, 2% DMSO) to 28% B over 95 minutes, then from 28% B to 40% B over 10 minutes, then from 40% B to 80% B over 7 minutes, the composition was held at 80% B for 3 minutes, and then dropped to 3% B over 0.1 minutes and held at 3% B for another 9 minutes. The Lumos™ Mass Spectrometer was operated in a stepped FAIMS data-dependent mode automatically switching between the acquisition of a single Orbitrap MS scan (120,000 resolution) every The identification of nanobody associated peptides and glycopeptides was accomplished using Byonic (Protein Metrics, version 3.9.6) (8). The MS raw files were searched with a MS1 tolerance of ± 5 ppm and a tolerance of ± 20 ppm for HCD / EThcD MS2 scans. Searches were performed using cysteine carbamidomethylation as a fixed modification, methionine oxidation as a variable modification in addition to allowing Nlinked glycosylation on asparagine residues. The default Byonic N-linked glycan database, which is composed of 309 mammalian N-glycans was used. The proteases specificity was set to full trypsin specificity and a maximum of two miss-cleavage events. Data was searched against the expected nanobody protein sequences. Searches was filtered to a 1% protein FDR as set in the Byonic parameters. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (9, 10) with the dataset identifier PXD023483.           Binding of the second WNbFc to RBD SARS-CoV-2 in the presence of the first WNbFc was calculated relative to the second WNbFc binding to RBD SARS-CoV-2 alone, which was assigned 100%. WNbFc fusions with more than 70% binding are considered non-competing and highlighted as green squares. (C) SEC analyses show that WNb 2 and WNb 10 binds to SARS-CoV-2 RBD simultaneously. The WNb 2 + WNb 10 + RBD complex elutes at a retention volume corresponding to higher molecular weight compared to the individual WNb 2 + RBD and the WNb 10 + RBD complexes on SEC. Excess of nanobodies elute as a second peak between retention volume of 14 to 16 mL.       X-ray diffraction data were collected on single crystals. * Values in parentheses are for highest-resolution shell.