Cell lines
HEK293T cells (ATCC, CRL-3216) and HeLa cells expressing hACE2 were kindly provided by Dr. Qiang Ding at Tsinghua University. Both of these cell lines were maintained at 37 °C in 5% CO2 in Dulbecco’s minimal essential medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin–streptomycin. FreeStyle 293 F cells (Thermo Fisher Scientific, R79007) were maintained at 37 °C in 5% CO2 in SMM 293-TII expression medium (Sino Biological, M293TII). Sf9 cells (ATCC) were maintained at 27 °C in Sf-900 II SFM medium. Hi5 cells (ATCC) were maintained at 27 °C in SIM HF medium.
Expression and purification of recombinant proteins
The genes encoding the ectodomain of S trimer and S2 trimer of prototype SARS-CoV-2 Wuhan-Hu-1 strain (GenBank: MN908947.3) were constructed as previously reported10. Both S trimer (residues M1-Q1208) and S2 trimer (residues S686-Q1208) contained proline substitutes at residue 986 and 987, a foldon trimerization motif, and a strep tag at C-terminal. Additional ‘GSAS’ substitution at furin cleavage site (residues 682-685) was also introduced into the S trimer construct to improve overall stability. Both S trimer and S2 trimer were expressed in the FreeStyle 293 F cells and purified by Strep-Tactin Sepharose (IBA Lifesciences 2-1201-025) followed by gel filtration chromatography (GE Healthcare). The gene encoding the ectodomain of SARS-CoV-2 Omicron BA.1 spike (GenBank ID: ULC25168.1) was constructed and contained the following modifications, proline substitutes at residue 817, 892, 899, 942, 986 and 987, “GSAS” substitutions at furin site, a foldon tag for trimerization, and a strep tag at C-terminal for purification. The trimeric Omicron BA.1 spike was expressed in the FreeStyle 293 F cells and purified by Strep-Tactin Sepharose followed by gel filtration chromatography. The S trimer of SARS-CoV-1, RBD of SARS-CoV-1, SARS-CoV-2 prototype and Beta strain, and the N-terminal peptidase domain of human ACE2 were expressed using the Bac-to-Bac Baculovirus Expression System (ThermoFisher 10359016) and Ni-NTA resin (QIAGEN 30230) followed by gel filtration chromatography, as previously reported78. Briefly, the RBD of SARS-CoV-2 prototype (residues Arg319–Lys529) with an N-terminal gp67 signal peptide for secretion and a C-terminal 6×His tag for purification was expressed using Hi5 cells and purified by Ni-NTA resin followed by gel filtration chromatography in HBS buffer (10 mM HEPES, pH 7.2, 150 mM NaCl). The RBD of SARS-CoV-1 (residues Arg306–Leu515) and N-terminal peptidase domain of human ACE2 (residues S19–D615) were expressed and purified by the same protocol as used for the RBD of SARS-CoV-2 prototype. The SARS-CoV-2 NTD protein was purchased from SinoBiological (40591-V49H).
Immunization of alpaca, construction of yeast display VHH library, and isolation of VHH yeasts specific for SARS-CoV-2 and SARS-CoV-1 spikes
The animal experiment protocol involving immunization, collection of blood samples, and construction of VHH library was approved by IACUC at NBbiolab, Inc. in Chengdu, China. The immunization procedure involved three-time subcutaneous injections of 200 μg recombinant RBD of prototype SARS-CoV-2 in Freund adjuvant, one-time subcutaneous injection of 1011 viral particle AdC68-19S vaccine expressing the prototype S trimer48, and two-time subcutaneous injections of 200 μg recombinant S-2P protein of prototype SARS-CoV-2 in Freund adjuvant. Seven days after the last immunization, blood samples were collected to isolate peripheral blood lymphocytes and plasma. Total RNA extracted from the peripheral blood lymphocytes was used as the template for the synthesis of the first strand cDNA, using oligo dT as a primer. VHH sequences were amplified by PCR, cloned into a yeast surface display vector pYD1, and transfected into the electrocompetent EBY100 cells. The yeast library was first grown in SDCAA media at 30 °C for 48 h. At the exponential growth phase, the yeast library was transferred to SGCAA media for induction of VHH expression at 20 °C for 36 h. The yeast clones displaying VHHs specific to SARS-CoV-2 or SARS-CoV-1 spikes were enriched by one round of MACS biopanning followed by an additional round of FACS biopanning. Specifically, the induced yeast library was collected and incubated with 100 nM SARS-CoV-2 S-2P protein or SARS-CoV-1 S protein on ice for 30 min. The yeast library was washed with cold PBS containing 1%FBS for three times and incubated with streptavidin microbeads on ice for 10 min. The mixture was passed through LS column and the S protein-positive yeasts were harvested for further culture and induction of VHH expression. Once again, induced yeasts were incubated with 100 nM SARS-CoV-2 S-2P protein or SARS-CoV-1 S protein on ice for 30 min. After extensive wash with cold PBS + 1%FBS, the yeast clones were incubated with HA-Tag (6E2) mouse monoclonal antibody conjugated with Alexa Fluor® 488 (Cell Signaling 2350 S, 1:100 dilution) and streptavidin conjugated with PE Conjugate (eBioscience 12-4317-87, 1:200 dilution) on ice for 30 min. The yeast clones were washed three times with cold PBS containing 1%FBS before analyzed by FACS using Aria II (BD Biosciences). Positive yeast clones were sorted and used for nanobody cloning.
Molecular cloning and expression of VHH
The sequences encoding various VHHs were amplified from the sorted yeast clones and inserted into two different expression vectors depending on the subsequent study objectives. One was to express VHH in conjunction with human IgG1 Fc fragment to evaluate binding and neutralizing activity of VHHs. The other was to express VHH with a 6×His tag for crystal structural analysis. For the former, VHH genes were cloned into the multiple cloning sites of pMD18T containing the upstream CMV promoter, the secretory signal sequence from the mouse Ig heavy chain, and the downstream human IgG1 Fc gene fragment and SV40 poly (A) signal sequence. For the latter, selected VHH genes were cloned into pVRC8400 vector with a 6×His tag. Expression and production of nanobodies were conducted by transfecting the expression vectors into the FreeStyle 293F cells using polyethyleneimine (PEI) (Polysciences 23966). After approximately 96 h, nanobodies containing the human IgG1 Fc in the culture supernatant were captured by AmMag Protein A Magnetic Beads (Genscript L00695) and eluted by 0.1 M Glycine pH 3.0. Nanobodies with 6×His tag were captured by Ni-NTA Agarose (QIAGEN 30230) and eluted by 500 nM Imidazole. All nanobodies were purified by gel-filtration chromatography with Superdex 200 High-Performance column (GE Healthcare). The exact concentration was determined by nanodrop 2000 Spectrophotometer (Thermo Scientific).
Production of SARS-CoV-2 and sarbecovirus pseudoviruses
Pseudoviruses carrying the full-length spike envelope of prototype SARS-CoV-2 and VOCs, and sarbecoviruses were generated as previously reported10. Specifically, human immunodeficiency virus backbones expressing firefly luciferase (pNL4-3-R-E-luciferase) and pcDNA3.1 vector encoding either SARS-CoV-2 or sarbecovirus spike proteins were co-transfected into the HEK293T cells (ATCC). Forty-eight hours later, pseudoviruses in the viral supernatant were collected, centrifuged to remove cell lysis, and stored at -80 °C until use. The wildtype pseudovirus used throughout the analysis was the prototype strain (GenBank: MN908947.3) with a D614G mutation (WT D614G). The Alpha variant (Pango lineage B.1.1.7, GISAID: EPI_ISL_601443) included a total of 9 reported mutations in the spike protein (69-70del, 144del, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H). The Beta variant (Pango lineage B.1.351, GISAID: EPI_ISL_700450) included 10 identified mutations in the spike such as L18F, D80A, D215G, 242-244del, S305T, K417N, E484K, N501Y, D614G and A701V. The Gamma variant (Pango lineage P.1, GISAID: EPI_ISL_792681) had 12 reported mutations in the spike including L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I and V1176F. The Delta variant (Pango lineage B.1.617.2, GISAID: EPI_ISL_1534938) included 10 reported mutations in the spike such as T19R, G142D, 156-157del, R158G, A222V, L452R, T478K, D614G, P681R, D950N. The Omicron BA.1 variant (Pango lineage BA.1, GISAID: EPI_ISL_6752027) was constructed with 34 mutations in the spike such as A67V, 69-70del, T95I, G142D, 143-145del, 211del, L212I, 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, and L981F. The Omicron BA.2 variant (Pango lineage BA.2, GISAID: EPI_ISL_8515362) was constructed with 29 mutations in the spike such as T19I, 24-26del, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, N969K and Q954H. The Omicron BA.2.12.1 variant (Pango lineage BA.2.12.1, GISAID: EPI_ISL_12560123) was constructed with 30 mutations in the spike such as T19I, 24-26del, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, N969K and Q954H. The Omicron BA.4/5 variant (Pango lineage BA.4/5, GISAID: EPI_ISL_12559461) was constructed with 30 mutations in the spike such as T19I, 24-26del, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, N969K and Q954H. The Kappa variant (Pango lineage B.1.617.1, GISAID: EPI_ISL_1384866) was constructed with 8 mutations in the spike such as T95I, G142D, E154L, L452R, E484Q, D614G, P681R and N1071H. The Eta variant (Pango lineage B.1.525, GISAID: EPI_ISL_ 2885901) included 8 mutations in the spike such as Q52R, A67V, 69-70del, Y144del, E484K, D614G, Q677H and F888L. The Iota variant (Pango lineage B.1.526, GISAID: EPI_ISL_ 2922249) was constructed with 6 mutations in the spike such as L5F, T95I, D253G, E484K, D614G and A701V. The Lambda variant (Pango lineage C.37, GISAID: EPI_ISL_ 2930541) was constructed with 8 mutations in the spike such as G75V, T76I, R246N, 247-253del, L452Q, F490S, D614G and T859N. The Epsilon variant (Pango lineage B.1.429, GISAID: EPI_ISL_ 2922315) was constructed with 4 mutations in the spike such as S13I, W152C, L452R and D614G. The variant with Pango lineage A23.1, GISAID: EPI_ISL_ 2690464) was constructed with 4 mutations in the spike such as F157L, V367F, Q613H and P681R. The G339D, S371L, S371F, S373P, T376A, R408S and S371L + D373P + S375F were constructed with a D614G mutation based on the WT strain. For sarbecoviruses, the cDNAs encoding the SARS-CoV-1 S glycoprotein (NCBI Accession NP_828851.1), Pangolin CoV GD spike (GenBank: QLR06867.1), Pangolin CoV GX spike (GenBank: QIA48614.1), Bat SARS-like coronavirus WIV16 (GenBank: ALK02457.1), Bat SARS-like RaTG13 spike (GenBank: QHR63300.2), Bat SARS-like ZC45 (GenBank: AVP78031.1), Bat SARS-like ZXC21 (GenBank: AVP78042.1) and Bat SARS-like coronavirus RsSHC014 (GenBank: AGZ48806.1) were synthesized with codons optimized for protein expression (Genwiz Inc., China) and verified by sequencing.
Neutralization activity of nanobodies against pseudoviruses and live SARS-CoV-2
Neutralizing activity of nanobodies were determined using SARS-CoV-2 pseudovirus and authentic live virus as previously reported55. Briefly, nanobodies were 3-fold serially diluted in 96-well cell culture plates, mixed with SARS-CoV-2 pseudovirus, and incubated at 37 °C for 1 h. HeLa-ACE2 cells were then added to the mixture of nanobody-pseudovirus, incubated at 37 °C for an additional 48 h, and lysed for measuring luciferase-activity. The IC50 values were calculated based on the reduction of 50% relative light units (Bright-Glo Luciferase Assay Vector System, Promega, E2650) compared to the virus-only control, using Prism 8.0 (GraphPad Software Inc., USA).
For the authentic live virus assay, we used the focus reduction neutralization test (FRNT) performed in a certified BSL3 facility at Shenzhen Third People’s Hospital, China. Briefly, serial dilutions of nanobodies were mixed with various authentic SARS-CoV-2 and incubated for 1 h at 37 °C. The mixtures were then transferred to 96-well plates seeded with Vero E6 cells and incubated for 1 h at 37 °C. After changing the medium, the plates were incubated at 37 °C for an additional 24 h. The cells were then fixed, permeabilized, and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG (SinoBiological 40588-T62, 1:1000 dilution) for 1 h at room temperature before adding Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG (Heavy chain + Light chain) antibody (TransGen Biotech HS101-01, 1:2000 dilution). The reactions were developed using KPL TrueBlue peroxidase substrate (Seracare Life Sciences 5510-0030). The number of SARS-CoV-2 foci was quantified using an EliSpot reader (Cellular Technology Ltd. USA). The authentic SARS-CoV-2 used in the assay included the WT, Alpha, Beta, Delta, and Omicron BA.1 and were isolated locally. Their whole genome sequences have been deposited into China National Center for Bioinformation, with accession numbers GWHXXXX00000000 for WT strain, GWHBFWX01000000 for Alpha strain, GWHBDSE01000000 for Beta strain, GWHBFWZ01000000 for Delta strain, and GWHBIRY01000000 for Omicron BA,1 strain, which are publicly accessible at https://ngdc.cncb.ac.cn/gwh. The viruses of Beta strain and Delta strain were gifted from Guangdong Provincial Center for Disease Control and Prevention, Guangdong Center for Human Pathogen Culture Collection (GDPCC).
Phylogenetic tree and genetic analysis of nanobodies
Genetic relatedness and clustering among isolated nanobodies were analyzed by neighbor-joining phylogenetic trees were generated using MEGA version 10.1.8 with 1000 bootstrap replicates. The IMGT/V-QUEST program (http://www.imgt.org/IMGT_vquest/vquest) was used to analyze the germline gene and the loop lengths of complementarity determining region 3 (CDR3) of each nanobody. Chord diagrams showing the germline gene usages and V/J gene pairing were analyzed and presented by the R package circlize version 0.4.1379. The width of the linking arc is proportional to the number of nanobodies identified. Sequence logo were plotted using Python package Logomaker80.
Nanobody competition with ACE2 and control antibody measured by SPR
The competitive binding to SARS-CoV-2 RBD between nanobodies and ACE2 and control antibody eCR3022.7 were analyzed using SPR (Biacore 8 K, GE Healthcare). Specifically, the prototype SARS-CoV-2 RBD was immobilized to a CM5 sensor chip (Cytiva BR100530) via the amine group for a final RU around 250. In the first round, nanobodies (1 μM) or eCR3022 (1 μM) were injected onto the chip for 120 s to reach the steady state for binding. In the second round, nanobodies (1 μM) or eCR3022 (1 μM) were injected onto the chip for 120 s followed by the injection of ACE2 (2 μM) or nanobodies (1 μM) for additional 120 s. In the third round, running buffer was injected for 120 s followed by injection of ACE2 (2 μM) or nanobodies (1 μM) for another 120 s. The sensorgrams of the three rounds were aligned from 120 to 240 s in Biacore 8 K Evaluation software (GE Healthcare). The blocking efficacy was determined by a comparison of the response units with and without prior antibody injection.
Western Blot analysis for proteinase-K resistance core
SARS-CoV-2 spike (residues M1-Q1208), containing a foldon trimerization motif and a strep tag at C-terminal were expressed in the FreeStyle 293 F cells and purified by Strep-Tactin Sepharose followed by gel filtration chromatography. 0.4 μg of the spike was incubated with 2 μg of the ACE2 or 2 μg of monomeric nanobodies for 1 h on ice. Trypsin (final concentration of 5 μg/mL, ThermoFisher 27250018) was then added to this mixture and incubated for an additional 20 min at 37 °C. Subsequently, the samples were subjected to proteinase-K (ThermoFisher AM2546) digestion with final concentration of 100 μg/mL and incubated 20 min at 37 °C. 4×SDS-PAGE loading buffer was then added to all samples prior to boiling at 100 °C. Samples were run on a 4–12% gradient Tris-MOPS-Gel (GenScript M00653) and transferred to polyvinylidene fluoride membranes. An anti-SARS-CoV-2 S2 polyclonal antibody (SinoBiological T40590-T62, 1:2000 dilution) and an HRP-conjugated anti-rabbit secondary antibody (Promega W4011, 1:4000 dilution) were used for Western blotting. The image was developed by AI600 and the intensity of proteinase-K resistant core was estimated by ImageJ 1.53k.
Cell surface staining
HEK 293 T cells were transfected with expression plasmids encoding SARS-CoV-2 prototype spike, and incubated at 37 °C for 24 h. Cells were digested from the plate with trypsin and distributed onto 96-well plates. Cells were washed twice with 200 µL staining buffer (PBS with 1% heated-inactivated fetal bovine serum (FBS)) between each of the following steps. First, cells were stained with each monomeric nanobody (20 μg/mL) at 4 °C for 20 min and then ACE2 (20 μg/mL) at 4 °C for 20 min. Anti-His tag-PE (Miltenyi Biotec 130-120-787, 1:200 dilution) and Streptavidin-APC (eBioscience 17-4317-82, 1:200 dilution) were added and incubated at 4 °C for 20 min. After extensive washes, the cells were resuspended and analyzed by BD LSRFortassa (BD Biosciences, USA) and FlowJo 10 software (FlowJo, USA). A human monoclonal antibody P1A-1D1, previously isolated by our group from SARS-CoV-2 convalescent individual and showed minimal competition with ACE255, was used as the antibody control and detected by staining with anti-human IgG Fc-PE (Biolegend 410708, 1:50 dilution). HEK 293 T cells with mock transfection were stained as background control. The number of positive cells in the selected gates were calculated (Fig. S4h).
Crystallization and data collection
To obtain the complex of RBD bound to nanobodies, RBD was incubated with each nanobody for 1 h on ice in HBS buffer. The mixture was then purified using gel filtration chromatography. Fractions containing the complex were pooled and concentrated to 10 mg/mL. Crystals were successfully grown at room temperature in sitting drops, over wells containing 0.2 M Ammonium sulfate, 0.1 M Bis-Tris pH 4.4, 21% w/v Polyethylene glycol 3,350 for Nb70-SARS-CoV-1 RBD, 0.15 M DL-Malic acid pH 7.0, 20% w/v Polyethylene glycol 3,350 for Nb70-1F11 Fab-SARS-CoV-2 RBD, 0.2 M Ammonium sulfate, 0.1 M Bis Tris pH 5.5, 25% w/v Polyethylene glycol 3,350 for 1-2C7-SA RBD, 0.2 M Ammonium formate, pH 6.6, 20% w/v Polyethylene glycol 3,350 for 3-2A2-4-SARS-CoV-2 RBD. Crystals were collected, soaked briefly in 0.2 M Ammonium sulfate, 0.1 M Bis-Tris pH 4.4, 21% w/v Polyethylene glycol 3,350 and 20% glycerol for Nb70-SARS-CoV-1 RBD, 0.15 M DL-Malic acid pH 7.0, 20% w/v Polyethylene glycol 3,350 and 20% glycerol for Nb70-1F11 Fab-SARS-CoV-2 RBD, 0.2 M Ammonium sulfate, 0.1 M Bis-Tris pH 5.5, 25% w/v Polyethylene glycol 3,350 and 20% glycerol for 1-2C7-SA RBD, 0.2 M Ammonium formate, pH 6.6, 20% w/v Polyethylene glycol 3,350 and 20% glycerol for 3-2A2-4-SARS-CoV-2 RBD and were subsequently flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K and a wavelength of 0.987 Å at the BL18U1 beam line for Nb70-SARS-CoV-1 RBD, Nb70-1F11 Fab-SARS-CoV-2 RBD and 1-2C7-SA RBD, 100 K and a wavelength of 1.07180 Å at the BL02U1 beam line for 3-2A2-4-SARS-CoV-2 RBD of the Shanghai Synchrotron Research Facility. Diffraction data were processed using the HKL3000 software (PMID:16855301) and the data-processing statistics are listed in Table S2.
Structure determination and refinement
The structure was determined using the molecular replacement method with PHASER in the CCP4 suite (PMID: 19461840). Density map improvement by updating and refinement of the atoms was performed with ARP/wARP26 (PMID: 18094467). Subsequent model building and refinement were performed using COOT (PMID: 15572765) and PHENIX (PMID: 12393927), respectively. Final Ramachandran statistics: 96.54% favored, 3.46% allowed and 0.00% outliers for the final Nb70-SARS-CoV-1 RBD structure, 97.28% favored, 2.59% allowed and 0.14% outliers for the final Nb70-1F11 Fab-SARS-CoV-2 RBD structure, 97.15% favored, 2.85% allowed and 0.00% outliers for the final 1-2C7-SA RBD structure and 93.89% favored, 5.79% allowed and 0.32% outliers for the final 3-2A2-4-SARS-CoV-2 RBD structure. The structure refinement statistics are listed in Table S2. All structure figures were generated with ChimeraX and Pymol (PMID: 28158668).
Conservation analysis of RBD sequences from SARS-CoV-2 and representative sarbecoviruses
SARS-CoV-2 spike sequences collected from December 2019 to June 2022 were obtained from GISAID EpiCoV database. Representative spike sequences from sarbecoviruses were obtained from NCBI Virus database. After filtration, over 8 million SARS-CoV-2 RBD sequences and 46 sarbecovirus RBD sequences were extracted. These RBD sequences were aligned using MAFFT v7.310 program. The entropy was calculated for each residue based on aligned sequences as follows:
$${{{{{\rm{Entropy}}}}}}=-\mathop{\sum }\limits_{i=1}^{21}p\left({x}_{i}\right)\log (p\left({x}_{i}\right))$$
(1)
In which xi are standard amino acids, plus gap. Figures were generated by UCSC ChimeraX 1.3.
Cryo-EM sample preparation and data collection
The trimeric spike of Omicron BA.1 at concentration of 1.5 mg/mL was incubated with 3-fold molar excess of 3-2A2-4 at room temperature for 1 hours. 4 μL of mixture was applied to glow-discharged holey carbon grids (Quantifoil grid, Cu 300 mesh, R1.2/1.3). The Quantifoil grids were blotted for 2 seconds with filter paper in 100% relative humidity at 8 °C and plunged into the liquid ethane to freeze samples using FEI Vitrobot system (FEI). Images for nanobody-spike trimer complexes were recorded using FEI Titan Krios microscope (Thermo Fisher Scientific) operating at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc.) at Tsinghua University. The automated software (AutoEMation2, PMID: 15797731) was used to collect 4795 movies for nanobody-spike trimer complexes in super-resolution mode at a nominal magnification of 81,000× with a pixel size of 1.0979 Å at a defocus range between -1.5 and-1.8 μm. Each movie has a total accumulate exposure of 50 e-/Å2 fractionated in 32 frames of 175 ms exposure.
Cryo-EM data processing
Motion Correction (MotionCor2 v.1.2.6, PMID: 28250466), CTF-estimation (GCTF v.1.18, PMID: 26592709), and non-templated particle picking (Gautomatch v.0.56, http://www.mrc-lmb.cam.ac.uk/kzhang/) were automatically executed by TsingTitan.py program. A total of 1,812,056 particles for nanobody-spike trimer complexes were extracted using RELION 3.0.8. Sequential data processing was carried out on cryoSPARC. After 2D classification, the best selected 947,196 particles were applied for initial model and 3D classification.
Nanobody protection against authentic SARS-CoV-2 infection in K18-hACE2 mice
Animal experiments were conducted in a Biosafety Level 3 (BSL-3) facility in accordance with the National University of Singapore (NUS) Institutional Animal Care and Use Committee (IACUC) (protocol no. R20-0504), and the NUS Institutional Biosafety Committee (IBC) and NUS Medicine BSL-3 Biosafety Committee (BBC) approved SOPs. Eight-week-old female K18-hACE2 transgenic mice (InVivos Ptd Ltd, Lim Chu Kang, Singapore) were used for this study. The mice were housed and acclimatized in an ABSL-3 facility for 72 h prior to the start of the experiment. K18-hACE2 transgenic mice were subjected to pretreatment of nanobody 3-2A2-4 (10 mg/kg) delivered through intraperitoneal injection a day prior to infection. The viral challenge was conducted through intranasal delivery in 25 μL of either 1.7 × 103 PFU of the infectious SARS-CoV-2 Omicron subvariant BA.1 or 103 PFU of Delta variant. Baseline body weights were measured prior to infection and monitored daily by two personnel post-infection for the duration of the experiment. Mice were euthanized when their body weight fell below 75% of their baseline body weight. To assess the viral load, mice from each experimental group were sacrificed 3 days post inoculation, with lung tissues harvested. Each organ was halved for the plaque assay and histology analysis, respectively. Tissues were homogenized with 0.5 mL DMEM supplemented with antibiotic and antimycotic (Gibco) and titrated in Vero E6 cells using plaque assays. Specifically, for virus titer determination, supernatants from homogenized tissues were serially diluted 10-fold in DMEM supplemented with antibiotic and antimycotic. Of each serial diluted supernatant, 250 μL was added to Vero E6 cells into 12-well plates. After 1 h of incubation for virus adsorption, the inoculum was removed and washed once with PBS. About 1.2% microcrystalline cellulose (MCC)-DMEM supplemented with antibiotic and antimycotic overlay media was added to each well and incubated at 37 °C, 5% CO2 for 72 h for plaque formation. The cells were then fixed in 10% formalin overnight and counterstained with crystal violet. The number of plaques was determined and the virus titers of individual samples were expressed in logarithm of PFU per organ. For histopathological analyses, lung lobes were fixed in 3.7% formaldehyde solution prior to removal from BSL-3 containment. The tissues were routinely processed, embedded in paraffin blocks (Leica Surgipath Paraplast), sectioned at 4 μm thickness, and stained with H&E (Thermo Scientific) following standard histological procedures. For immunohistochemistry, sections were deparaffinized and rehydrated, followed by heat-mediated antigen retrieval, quenching of endogenous peroxidases and protein blocking. Sections were then covered with rabbit anti-SARS-CoV-2 N protein monoclonal antibody (Abcam ab271180, 1:1000 dilution) for 1 h at room temperature. Subsequently, sections were incubated with rabbit-specific HRP polymer (secondary antibody), visualized using chromogenic substrate DAB solution (Abcam), and counterstained with hematoxylin.
Statical analysis
The technical and independent experiment replicates were indicated in the figure legends. Half-maximal inhibitory concentration (IC50) of nanobodies was calculated by the equation of four-parameter dose inhibition response using Graphpad Prism 8.0. The fold change of the variants relative to D614G in neutralization was calculated by simple division of respective IC50 values. In animal experiments, a two-tailed unpaired Mann-Whitney test was used to assess statistical significance. Statistical calculations were performed in GraphPad Prism 8.0. Differences with p-values less than 0.05 were considered to be statistically significant (**p < 0.01).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
