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[purple nature background]Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants

Time:2021-09-17 19:06:07

  No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

  To replace the entire mouse VH locus (mm10, chromosome 12: 113,567,224–116,010,427) we assembled a targeting vector (pLH28-exchange-cassette) and a VHH minigene. The targeting vector was built by inserting a selection cassette composed of pEF1a-Puro-TK-2A-EGFP in between wtLoxP and LoxP257 sites within the pLH28 exchange vector24. As homology arms, 1-kb and 0.8-kb fragments flanking the VH deletion domain were cloned 5′ and 3′ of the LoxP sites. To build the VHH minigene, VHH genes (18 from alpaca, 7 from dromedary and 5 from Bactrian camel) were selected on the basis of published sequences25,26,27. Thirty mouse VH promoters (250 bp) were next chosen on the basis of their expression as measured by GRO-seq in resting and activated mouse B cells. VHHs were codon-optimized and complemented with mouse leading exons, introns and recombination signal sequences. The 30 units were pieced together by Gibson assembly (NEB) into the pBeloBAC11 vector.

  E14 cells were cultured in Glasgow’s MEM (Thermo Fisher Scientific, 11710035) supplemented with 10% FBS (ATCC, SCRR-03-2020), Glutamax, sodium pyruvate, non-essential amino acids (NEAA), penicillin (50 units per ml)–streptomycin (50 μg ml?1) and β-mercaptoethanol (Thermo Fisher Scientific, 35050061, 11360070, 11140050, 15140122, 21985023, respectively) at 37?°C and 5% CO2. mLIF (GeminiBio, 400-495, 10,000×), MEKi (Stemgent, 0400602, 10,000×) and GSKi (Stemgent, 0400402, 3,333×) were added to the medium before use. Dishes and plates were coated with 0.1% glycine for 15 min at room temperature before use. To delete the CH1 exon of Cμ, sgRNAs targeting the flanking introns were cloned into the CRISPR–Cas9 plasmid pX458 (Addgene, 48138). A 100-nt-long single-stranded oligonucleotide (ODN) donor (100 μM, 3 μl) was co-transfected with the two Cas9 sgRNA plasmids (2 μg each) into embryonic stem (ES) cells (2 million cells) with the Amaxa nucleofection kit (Lonza, VPH-1001, programme A030). After 24 h of culture, GFPhigh ES cells were FACS-sorted and cultured in 10-cm dishes at a concentration of 2,000 cells per dish. Seven days later, colonies were transferred into 96-well plates and cultured for an additional 3 days. Genomic DNA was then extracted and genotyped for Cμ exon deletion. Clones with homozygous deletions were selected to next delete the Cγ1 exon with the same strategy. To delete the entire VH locus, sgRNAs targeting sequences upstream of Ighv1-86 (first Ighv) and downstream of Ighv5-1 (last Ighv), respectively, were cloned into pX458. Selected ES cells (2 million cells) were co-transfected with the two Cas9 sgRNA plasmids (1.5 μg each) and the pLH28-exchange-cassette plasmid (1.5 μg) and then cultured in 10-cm dishes. Twenty-four h later, cells were selected with puromycin (0.8 μg ml?1) for 10 days and individual colonies were picked for expansion and genotyping by long-range PCR. Positive clones (2 million cells) were co-transfected with VHH minigene vector (3 μg) and a Cre-expressing plasmid (1 μg) and cultured in 10-cm dish for 3 days. Cells were then selected with ganciclovir (2 μg ml?1) for 7 days before individual colonies were picked for expansion and genotyping. sgRNAs and ODN primers are listed in Supplementary Table 4.

  Two modified ES cell clones with normal karyotype were injected into C57BL/6 blastocysts, which were then transferred to the uteri of pseudopregnant C56BL/6 recipients. High-percentage chimeras were mated to C57BL/6 mice and offspring were genotyped for VHH minigene knock-in and Cμ and Cγ1 exon deletion. One out of three chimeras produced F1 offspring that showed germline transmission. Three F1 male mice were backcrossed with C57BL/6 mice. F2 heterozygous mice were inbred to produce mice homozygous for all three modifications. Two F1 offspring from the same chimera were null for Ch1 of Ighm but wild type for VH and CH1 exon of Cγ1. These mice were used as controls for Extended Data Fig. 3b.

  B cells were activated by culturing them in RPMI 1640 supplemented with 10% FBS, HEPES, sodium pyruvate, NEAA, penicillin–streptomycin and β-mercaptoethanol at 37?°C and 5% CO2 in the presence of lipopolysaccharide (LPS,?Sigma, L2630, 50 μg ml?1), interleukin-4 (IL-4, Sigma, I1020, 2.5 ng ml?1) and anti-CD180 (1:2,000, BD Pharmingen, 552128) antibody for 72 h. For proliferation assays, cells were stained with CellTracer Violet (Thermo Fisher Scientific, C34557) at room temperature for 20 min before culturing for 96 h. For all FACS staining, cells were incubated in FACS buffer (phosphate-buffered saline (PBS), 2% FBS) at 4?°C for 20 min. Antibodies used for staining were: anti-B220-PerCP–Cy5.5 (1:500, eBioscience, 45-045-82), anti-B220–APC (1:500, Invitrogen, 17-0452-83), anti-IgM–APC (1:500, eBioscience, 17-5790-82), anti-Igκ–PE (1:500, BD Pharmingen, 559940), anti-Igκ–FITC (1:500, BD Pharmingen, 550003), anti-Igλ–FITC (1:200, BD Pharmingen, 553434), anti-IgG1–PE (1:200, BD Pharmingen, 550083), anti-IgG1–APC (1:200, BD Pharmingen, 550874), anti-IgD–FITC (1:200, BD Pharmingen, 553439), anti-CD95–PE (1:200, BD Pharmingen, 554258), anti-CD43–PE (1:200, BD Pharmingen, 553271), anti-CD23–PE (1:200, BD Pharmingen, 553139), anti–CD21-FITC (1:200, Biolegend, 123408) and Viability Dye eFluor506 (1:1000, Invitrogen, 1923275). Data were acquired using BD FACSCanto and FACSDiva software and analysed with FlowJo software. Gating strategy is shown in Extended Data Fig. 10d.

  Genomic DNA from bone marrow or splenic samples was extracted with the DNeasy Blood & Tissue kit (Qiagen, 69506). VHH(D)J joints were PCR-amplified from 100 ng of DNA with a framework primer unique for each of the 30 VHHs, and a common downstream JH4 primer. PCR products were loaded onto 1% agarose gel to resolved them by size. Primers are listed in Supplementary Table 4.

  VHH(D)J phagemid libraries from unimmunized mice were constructed by first extracting RNA from nanomouse splenic samples with Trizol (Thermo Fisher Scientific, 15596026) and reverse-transcribed to cDNA with SuperScript III (Thermo Fisher Scientific, 18080400) according to the manufacturer’s instructions with some modifications. Ten μg of total RNA was denatured and annealed with gene-specific primers corresponding to CH2 of Ighm. After elongation at 50?°C for 50 min, template-switching oligonucleotide (TSO) (3′-propyl modified) linker was added to the?reaction and the first strand cDNA was elongated for another 90 min at 42?°C. The reaction was inactivated at 85?°C for 5 min and 2 μl of cDNA was used as template for VHH(D)J amplification by two-step PCR with HiFi PCR Premix (Takara, 639298). For the first-step PCR, unmodified TSO and Ighm-CH2-specific oligonucleotides were used. Thirty ng of the first-step PCR product was then amplified with a primer mix of 30 forward primers corresponding to framework (FR1) of 30 VHH genes and 4 reverse primers corresponding to JH1~JH4. pMES4 phagemid (Addgene, 98223) was amplified with primers to introduce SfiI sites on both ends. VHH(D)J and pMES4 fragments were then digested with SfiI (NEB, R0123L) and ligated (100 and 200 ng, respectively) with T4 ligase (NEB, M0202L) at 16?°C overnight. Ligation product was purified with DNA Clean & Concentrator (Zymo Research, D4014) and eluted into 12 μl of water. Three μl of DNA was electroporated into 60 μl of TG1 cells (Lucigen, 60502-2) in 1.0-mm cuvette (HARVARD Apparatus, 450134) with BTX electroporation system ECM 630 at the setting of 25 μF, 200 ohms, 1,600 volts. After 1 h recovery in 37?°C in shaker incubator, TG1 cells were plated on 5 of 10-cm LB agar plates supplemented with 100 μg ml?1 carbenicillin (KD Medical, BPL-2400). Plates were placed in 37-°C bacteria incubator overnight, bacteria were scraped off plates and phagemid library was DNA-extracted with Zymo Plasmid Miniprep kit (Zymo Research, D4054). Primers are listed in Supplementary Table 4.

  VHH(D)J recombinants from splenic cells of two nanomice were PCR-amplified as described in ‘VHH(D)J recombinants phagemid library construction’, and then cloned directly into pCR-Blunt II-TOPO vector (Thermo Fisher Scientific, 450245) and transformed into Stabl3 competent Escherichia coli (Thermo Fisher Scientific, C737303). Ninety-six colonies were randomly picked for Sanger sequencing. TG1 cells from BG505 DS-SOSIP immunized nanomouse phagemid library were plated onto carbenicillin-containing plates and 50 colonies picked for Sanger sequencing. Sequence alignment was performed using Snapgene software.

  All animal-related procedures were performed by following our NIAMS ACUC protocol. To monitor the germinal centre reaction, three nanomice and two C57BL/6 mice were immunized intraperitoneally with 50 μg of keyhole limpet haemocyanin (KLH) in the presence of complete Freund’s adjuvant (CFA). A boost injection was performed in the footpads with 25 μg of KLH in the presence of incomplete Freund’s adjuvant (IFA) on day 6. Spleen samples were collected on day 12 for analysis.

  To isolate nanobodies recognizing HIV-1 envelope trimer, one nanomouse was immunized intraperitoneally with 50 μg of BG505 DS-SOSIP in the presence of CFA on day 0, and boost immunized with 25 μg of BG505 DS-SOSIP in the presence of IFA or PBS on day 22 and 44, respectively. Bone marrow, spleen and blood were collected on day 48.

  To isolate neutralizing nanobodies against SARS-CoV-2, a llama (Capralogics) was immunized subcutaneously with 1 mg of recombinant RBD protein in the presence of CFA at day 0, and boost immunized with 0.5 mg of RBD protein in the presence of IFA on days 14, 28 and 42. Two more boost immunizations with 0.5 mg of recombinant spike protein in the presence of IFA were performed on day 56 and 70. On day 80, 500 ml of whole blood were collected for library preparation.

  To isolate SARS-CoV-2 neutralizing nanobodies from nanomice, two groups of mice (five for group 1 and six for group 2) were immunized with RBD and/or spike protein and bleeds were collected after a 62-day immunization protocol. Mice were immunized intraperitoneally with 50 μg of RBD protein (group 1) or spike protein (group 2) in the presence of CFA on day 0, and boost immunized intraperitoneally with 25 μg of RBD protein (group 1) or spike protein (group 2) in the presence of IFA on days 14, 28 and 42. Mice were further immunized with 25 μg of spike protein in PBS on day 56 and 59, intraperitoneally and intravenously, respectively. Bone marrow, spleen and blood samples were collected on day 62. The best responders—nanomouse 1 (group 1) and nanomice 2 and 3 (group 2)—were selected for phage library construction.

  The llama phage library was constructed as previously described28 with some modifications. In brief, 300 ml of whole blood was collected from llama and peripheral blood mononuclear cells (PBMCs) were enriched using Ficoll-Paque plus (GE Healthcare, 17-1440-03). Fifty μg of extracted RNA was reverse-transcribed to cDNA with random hexamers and 2.5 μl of cDNA was used for first round RT–PCR with gene-specific primers CALL001 and CALL002. The PCR reaction was repeated in 12 individual tubes with cDNA added into reactions separately. PCR fragments of about 700-bp long were gel-purified and used as template (30 ng for each reaction, repeated in 12 individual tubes) for second round PCR with nested primers VHH-Back and VHH-For. PCR product from individual reactions were pooled and gel-purified. Nanobody fragments and pMES4 phagemid were digested with PstI-HF and BstEII-HF restriction enzymes (NEB: R3140L, R3162L) and ligated (1 μg and 2 μg respectively) with T4 ligase at 16?°C overnight. Ligation product was column-purified (into 12 μl of H2O) and electroporated into 360 μl of TG1 cells. After 1 h of recovery at 37?°C in a shaker incubator, cells were plated on 6 of 245 × 245-mm dish (Thermo Fisher Scientific, 431301) containing 2-YT agar supplemented with 100 μg ml?1 carbenicillin and 2% (w/v) glucose. Plates were placed in a 37-°C bacteria incubator overnight and then bacteria were scraped off of plates and archived as glycerol stocks. Cells were infected with VCSM13 helper phage (Agilent Technologies, 200251) followed by precipitation of culture supernatant with 20% polyethylene glycol 8000 (Sigma, 89510) in 2.5 M sodium chloride on ice to purify the nanobody phage particles. Phage particles were resuspended in 1 ml PBS, 300 μl were used for screening immediately and the remaining phages were stored at ?80?°C in the presence of 10% glycerol.

  Nanomouse nanobody phage libraries were constructed the same way as nanomouse VHH(D)J region phagemid library construction with some modifications. In brief, total RNA was extracted from splenic cells, bone marrow cells and PBMCs of immunized mice and processed separately until the TG1 cell electroporation step. RNA from splenic cells, bone marrow and PBMCs (50 μg, 50 μg and all, respectively) were reversed-transcribed to cDNA with Ighg1-CH2-specific primer in separate tubes. Two μl of cDNA was used as template for PCR variable domain amplification (12 reactions each), using unmodified TSO and Ighg1-CH2-specific oligonucleotide as primers. Second PCR was repeated in 12 reactions using 30 ng of the first-step PCR product as template and 30 FR1 and 4 JH oligonucleotide mix as primers. PCR products were gel-purified, digested with SfiI and ligated with pMES4 (200 ng and 400 ng, respectively). Ligation products from splenic cells, bone marrow and PBMC samples were pooled and column-purified (into 12 μl of water) and electroporated into 360 μl of TG1 cells and phage libraries prepared as described in ‘VHH(D)J recombinants phagemid library construction’. Primers are listed in Supplementary Table 4.

  Phagemid DNA extracted from TG1 cell libraries was used as starting material for constructing MiSeq libraries to measure VHH use and nanobody diversity. In brief, 1.2 μg of phagemid DNA was used as template and VHH(D)J inserts were amplified with primers recognizing the pMES4 backbone using CloneAmp HiFi PCR Premix (Takara, 639298) in a 50-μl reaction (9 cycles). To avoid MiSeq failure owing to low complexity at initial cycles and to enable multiplex sequencing, 1–9-nt-long staggers were introduced into forward primers. Without purification, 5 μl of the first PCR product were used as template for a second PCR (9 cycles) to add Illumina P5 and P7 primers on both ends. PCR product was then loaded onto a 2% agarose gel and the approximately 580-bp size band was purified with Zymoclean Gel DNA Recovery Kit (Zymo Research, D4002). DNA concentration was determined by Qubit 4 Fluorometer (Thermo Fisher Scientific, Q33238) and average DNA size was determined by TapeStation 4150 (Agilent). DNA was then adjusted to 2 nM in elution buffer containing 0.1% Tween-20. For unimmunized nanomice VHH(D)J library, DNA (2 nM) from 3 mice was mixed at a 1:1:1 ratio and loaded for MiSeq run. For immunized llama and nanomice nanobody diversity analysis, DNA (2 nM) of pre-selection and post-selection libraries were mixed at 10:1 ratio first and then samples from individual animals were pooled at 1:1 ratio before loading for MiSeq sequencing. Primers are listed in Supplementary Table 4.

  For unimmunized nanomouse VHH use analysis, pooled library from 3 mice was sequenced by MiSeq (pair end, 270 cycles × 2). Pair-end reads were merged with NGmerge29 with default settings. Nucleotides corresponding to pMES4 were trimmed using pTrimmer program30, leaving clean VHH(D)J sequences in the merged reads. Reads with undetermined N nucleotides, low quality sequence or less than 300 nt in length were removed with the fastp program31. Fastq format sequences were converted to .fasta format for further analysis. To calculate VHH use, a BLAST database was built from a .fasta format file (vhh.exon.fa) containing the exon sequence of all 30 VHH genes, using BLAST+. VHH(D)Js were then aligned to VHH genes using igblast program32. The alignment output file was simplified to retain only sequence identifier and VHH(D)J recombination information.

  For immunized llama and nanomouse nanobody diversity analysis, in total 8 libraries (pre- and post-selection) were sequenced by MiSeq (pair end, 300 cycles × 2). The 3′-end low-quality sequences were trimmed using the Sickle program (v1.33, available at https://github.com/najoshi/sickle). For different libraries, the minimum length of trimmed sequence was adjusted on the basis of the length of staggers in the primers used for library construction. Paired sequences were merged by flash program (v.1.2.11)33 and translated. To extract nanobody sequences and to locate CDR3 regions, we used ANARCI program34 to annotate VHH genes with IMGT numbering. Protein sequences with greater than or equal to 100 amino acids in total and greater than or equal to 1 amino acid in the CDR3 region were extracted for further analysis. Enrichment of individual sequences were calculated by comparing their frequencies in pre- and post-selection libraries. Sequences that were enriched more than 10 times and had greater than or equal to 5 × 10?5 frequency were selected for CDR3 clustering using cd-hit program (v.4.6.8)35.

  BG505 DS-SOSIP protein was expressed and purified as previously described36. The spike protein of SARS-CoV-2 and its RBD were expressed and purified as previously described37,38 with some modifications. In brief, 1 mg of pCAGGS-Spike or pCAGGS-RBD plasmid was transfected into 1 l of Expi293 cells (Thermo Fisher Scientific, A14528) with Turbo293 transfection reagent (Speed Biosystem, PXX1002). Supernatants from transfected cells were collected on day 4 after transfection by centrifugation of the culture at 12,000g for 15 min. Supernatant was then filtered through 0.2 m aPES filter (Thermo Fisher Scientific, 5670020) and incubated with 10 ml of cOmplete His-tag purification resin (Roche, 05893801001) for 1 h at room temperature. Next, His-tag resin was collected through gravity flow columns (BioRad, 9704652), washed with 100 ml of washing buffer (15 mM imidazole, 50 mM TrisHCl, 300 mM NaCl) and eluted with 25 ml of elution buffer (300 mM imidazole, 50 mM TrisHCl, 300 mM NaCl). Eluate was concentrated in 10-kDa Amicon Centrifugal Units (EMD Millipore, UFC901024) and then dialysed in PBS using Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, 66381). Proteins were analysed by NuPAGE gel (Thermo Fisher Scientific, NP0336BOX) and visualized by InstantBlue staining (Abcam, ab119211). Soluble spike trimers or monomeric RBD proteins were aliquoted, snap-frozen by liquid nitrogen and stored at ?80?°C before being used for immunization. RBD and spike (HexaPro) proteins used for phage screening, BLI, negative-stain electron microscopy and cryo-electron microscopy (cryo-EM) were done as previously described10,39.

  RBD, spike and BG505 DS-SOSIP were coated by different methods onto MaxiSorp 96-well plate (Thermo Fisher Scientific, 439454) for phage screening. For RBD screening, two wells were coated with 50 μl of RBD protein (100 μg ml?1 in PBS) at 4?°C overnight. Another well with 50 μl of PBS was used as an non-coated control. Wells were washed with PBS with 0.1% Tween-20 three times and blocked with 5% non-fat milk in PBS at room temperature for 1 h. For spike or BG505 DS-SOSIP screening, three wells were coated with 50 μl of lectin (EMD Millipore, L8275, 100 μg ml?1 in PBS) at 4?°C overnight. Wells were washed and blocked with 10% non-fat milk in room temperature for 1 h. After three washes, 50 μl of 100 μg ml?1 of BG505 DS-SOSIP or spike were added to 2 wells, incubated at room temperature for 2 h and washed. A third well contained PBS and served as a non-coated control. Three hundred μl of phage particles was mixed with 300 μl of 10% non-fat milk and rotated gently at room temperature for 1 h. One hundred and fifty μl of blocked phage particles was then added into each well and incubated in room temperature for 2 h with gentle shaking. After 15 washes, phages were eluted with TrypLE Express Enzyme (Thermo Fisher Scientific, 12605010) by shaking plates at 700 rpm at room temperature for 30 min and used immediately for selection efficiency estimation (10 μl of phage eluate) and recovery infection (the remaining eluate) as previously described28. Anti-RBD libraries were selected with RBD protein once, and libraries constructed from BG505 DS-SOSIP or spike immunized animals were selected with BG505 DS-SOSIP or spike (HexaPro) proteins twice.

  After one or two rounds of selection, recovered TG-1 cells were plated and colonies were picked to prepare periplasmic extracts containing crude nanobodies for enzyme-linked immunosorbent assay (ELISA). In brief, individual colonies were picked and grown in 96 deep-well plates (Thermo Fisher Scientific, 278743) in 2YT medium supplemented with 100 μg ml?1 of carbenicillin and 0.1% glucose. IPTG (final 1 mM) was added when optical density at 600 nm (OD600) reached 1 and protein expression was induced in 30?°C for 16 h. Periplasmic extracts were prepared by resuspending bacteria pellet in 200 μl of PBS and rapidly frozen in liquid nitrogen. Frozen cells were thawed slowly at room temperature and centrifuged at 4,100g for 15 min. Maxisorp plates were coated with lectin (2 μg ml?1) followed by BG505 DS-SOSIP (2 μg ml?1) or with RBD (2 μg ml?1). After blocking, 100 μl of nanobody-containing supernatant were transferred to the plates and incubated for 2 h at room temperature. Plates were washed and then incubated with horse radish peroxidase (HRP) conjugated goat anti-alpaca VHH domain specific antibody (Jackson ImmunoResearch, 128-035-232) for 1 h at room temperature. Plates were washed and then developed by addition of 50 μl of tetramethylbenzidine (TMB) (Thermo Fisher Scientific, 34028) for 10 min, then the reaction was stopped by adding 50 μl of 1 M H2SO4. Absorbance at 450 nm was measured with Synergy microplate reader (BioTek Gen5).

  Phagemids from lead candidates identified by ELISA were extracted from TG-1 cells and transformed into WK6 cells (ATCC, 47078). Cultures were grown in 30 ml of 2YT medium (100 μg ml?1 of carbenicillin and 0.1% glucose) at 37?°C and 220 rpm until OD600 reached 1. Protein expression was induced by 1 mM IPTG at 30?°C for 16 h and then pelleted at 4,100g for 15 min. The resulting pellets were resuspended in 1 ml of PBS plus 30 μl of 0.5 MU ml?1 polymyxin B (Sigma, P1004) and incubated at 37?°C with shaking for 1 h. Cell debris were pelleted at 12,000g for 5 min and nanobodies in the supernatant were purified using Capturem His-tagged purification kit (Takara, 635710). For larger-scale nanobody production (0.2 to 1 l of culture), nanobodies in the supernatant were purified by cOmplete His-tag purification resin and dialysed in PBS as described in ‘Expression and purification of BG505 DS-SOSIP and SARS-CoV-2 proteins’. Proteins were filtered sterile by 0.22-μm PVDF membrane (EMD Millipore, UFC30GVNB) before being used for downstream assays.

  Monomeric or trimeric nanobody sequences were fused to the Fc region of human IgG1 with 6×His tag at the C-terminal end and cloned into the pVRC8400 vector. In trimeric form, nanobody units were connected through (GGGGS)×3 flexible linkers. In some cases, llama IgG2a hinge region was used in lieu of human IgG1 hinge. The Fc fusion constructs were expressed in Expi293 cells as described in ‘Expression and purification of BG505 DS-SOSIP and SARS-CoV-2 proteins’ at 33?°C from day 2 to day 4. Antibodies in the supernatant were purified using either cOmplete His-tag purification resin or protein A (Thermo Fisher Scientific, A26457). When protein A resin was used, antibodies were eluted by IgG elution buffer (Thermo Fisher Scientific, 21009) and brought to neutral pH by adding 1/10 volume of Tris-HCl (1M, pH 8). Antibodies were concentrated, dialysed and filtered. Nanobody–Fc yields were up to 100 mg l?1. RBD region of SARS-CoV, SARS-CoV-2 and bat coronavirus WIV16 spike protein were fused to the Fc region of human IgG1 and cloned into pVRC8400 vector. RBD–Fc proteins were expressed in Expi293 cells and purified with protein A.

  RBD–ACE2 interaction blocking potential of nanobodies was tested using the SARS-CoV-2 surrogate virus neutralization test (sVNT) kit (Genscript, L00847) according to the manufacturer’s instructions. In brief, HRP–RBD was diluted and incubated with specified concentrations of nanobodies for 30 min at 37?°C. Samples were then transferred onto ACE2-coated plates and incubated for 15 min at 37?°C. Plates were washed, and the assay was developed using TMB reagent and quenched with stop solution. Absorbance at 450 nm was measured with a Synergy microplate reader (BioTek Gen5). Inhibition rate was calculated and plotted using Microsoft Excel according to manufacturer’s instruction of the sVNT kit.

  A panel of plasmids expressing RBD-mutant SARS-CoV-2 spike proteins in the context of pSARS-CoV-2-SD19 have previously been described1,40. The mutants E484K and KEN (K417N, E484K and N501Y) were constructed in the context of a pSARS-CoV-2-SΔ19 variant with a substitution in the furin cleavage site (R683G). The IC50 of these pseudotypes were compared to a wild-type SARS-CoV-2 spike sequence carrying R683G in the subsequent analyses, as appropriate. Generation of SARS-CoV-2 pseudotyped HIV-1 particles and pseudovirus neutralization assay was performed as previously described11. In brief, 293T cells were transfected with pNL4-3DEnv-nanoluc and pSARS-CoV-2-SD19 and pseudotyped virus stocks were collected 48 h after transfection, filtered and stored at ?80?°C. Serially diluted nanobodies were incubated with the pseudotyped virus for 1 h at 37?°C. The mixture was added to 293TACE211 (for analysis of wild-type neutralization activity) (Fig. 2) or HT1080Ace2 cl.1417 cells (for analysis of spike mutant panel) (Fig. 3), and after 48 h cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5x reagent (Promega). Nanoluc luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega) with Modulus II Microplate Reader User interface (TURNER BioSystems). The relative luminescence units were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of antibodies. Neutralization of HIV-1-based SARS-CoV-1 and bat coronavirus WIV16 pseudotypes were performed in HT1080/ACE2cl.14 cells as previously described41. The IC50 for nanobodies was determined using four-parameter nonlinear regression (GraphPad Prism).

  Recombinant Indiana vesicular stomatitis virus (rVSV) expressing different coronavirus spikes (SARS-CoV-2, RaTG13, GDPangolin, GXPangolin, SARS-CoV, WIV1, SHC014, LYRa11, Rs7327, Rs4084 and Rs4231) were generated as previously described2,42,43. In brief, HEK293T cells were grown to 80% confluency before transfection with the spike gene using Lipofectamine 3000 (Invitrogen). Cells were cultured overnight at 37?°C with 5% CO2, and VSV-G pseudo-typed ΔG-luciferase (G*ΔG-luciferase, Kerafast) was used to infect the cells in DMEM at a multiplicity of infection (MOI) of 3 for 2?h before washing the cells with 1× DPBS three times. The next day, the transfection supernatant was collected and clarified by centrifugation at 300g for 10?min. Each viral stock was then incubated with 20% I1 hybridoma (anti-VSV-G, ATCC: CRL-2700) supernatant for 1 h at 37?°C to neutralize contaminating VSV-G pseudo-typed ΔG-luciferase virus before measuring titres and making aliquots to be stored at ?80?°C. Neutralization assays were performed by incubating pseudoviruses with serial dilutions of antibodies and scored by the reduction in luciferase gene expression as previously described2,42,43. In brief, 293TACE2 cells were seeded in 96-well plates (2?×?104 cells per well). Pseudoviruses were incubated with serial dilutions of the antibodies in triplicate for 30 min at 37?°C. The mixture was added to cultured cells and incubated for an additional 16 h. Luminescence was measured using Luciferase Assay System (Promega), and IC50 was defined as the dilution at which the relative light units were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background in the control groups with cells only. The IC50 values were calculated using a five-parameter dose–response curve in GraphPad Prism.

  The SARS-CoV-2 viruses USA-WA1/2020 (WA1), USA/CA_CDC_5574/2020 (B.1.1.7), hCoV-19/South Africa/KRISP-EC-K005321/2020 (B.1.351) and hCoV-19/Japan/TY7-503/2021 (P.1) were obtained from BEI Resources (NIAID, NIH) and propagated for one passage using Vero E6 cells. Virus infectious titre was determined by an end-point dilution and cytopathic effect (CPE) assay on Vero E6 cells as previously described42. An end-point dilution microplate neutralization assay was performed to measure the neutralization activity of nanobodies. In brief, nanobodies were subjected to successive fivefold dilutions starting from 10 μg ml?1. Triplicates of each dilution were incubated with SARS-CoV-2 at an MOI of 0.1 in EMEM with 7.5% inactivated fetal calf serum for 1 h at 37?°C. After incubation, the virus–nanobody mixture was transferred onto a monolayer of Vero E6 cells grown overnight. The cells were incubated with the mixture for about 70 h. CPE of viral infection was visually scored for each well in a blinded fashion by two independent observers. The results were then reported as percentage of neutralization at a given nanobody dilution. The IC50 for nanobodies was determined using nonlinear regression (normalized response, variable slope) in GraphPad Prism.

  Nanobody was nebulized with a portable mesh nebulizer (Philips, InnoSpire Go) producing 2-5 μm particles at a final concentration of 0.4 mg ml?1. The resulting aerosol was collected by condensation into a 50-ml tube cooled on ice. Pre- and post-nebulization samples were analysed by NuPAGE gel and visualized by InstantBlue staining. SARS-CoV-2 surrogate virus neutralization test was also performed to compare the neutralization potency of pre- and post-nebulization samples. For thermostability tests, nanobodies supplemented with loading buffer (Thermo Fisher Scientifc, NP0007) and β-mercaptoethanol were heated at 98?°C for 10 min and then analysed on a NuPAGE gel and visualized by InstantBlue staining.

  The BLI assay was performed using a fortéBio Octet Red384 instrument to determine the affinity of nanobodies to RBD. In brief, biotinylated-RBD was immobilized onto streptavidin-coated biosensors and then dipped into a solution containing the nanobody for 30 s followed by dissociation for 2–3 min. To assay the binding of nanobodies to RBD–Fc (SARS-CoV, SARS-CoV-2 and WIV16), 6×His-tagged nanobody was immobilized onto Ni-NTA coated biosensors and then dip into RBD–Fc solution for association for 1 min followed by dissociation for 1 min. Sensorgrams of the concentration series were corrected with corresponding blank curves and fitted globally with Octet evaluation software using a 1:1 Langmuir model of binding.

  Nanobody–RBD binding competition assay was performed using a fortéBio Octet Red384 instrument. Biotinylated-RBD was first immobilized onto streptavidin coated biosensors and allowed to associate with one of the six nanobodies, then dipped into a solution contained a second nanobody.

  Nanobody–spike complexes were prepared by mixing the two proteins at a 1:1 weight ratio, then diluted with a buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, adsorbed to a freshly glow-discharged carbon-coated copper grid, washed with the above buffer, and stained with 0.75% uranyl formate. Images were collected at a magnification of 57,000 using EPU on a Thermo Fisher Talos F200C microscope equipped with a 4k × 4k CETA 16 M camera and operated at 200 kV. The pixel size was 2.5 ? for the CETA camera. Particle picking, reference-free 2D classification, 3D reconstruction and refinement were performed using cryoSPARC.

  Nanobody–spike complexes (Nb12–S6P and Nb30–S6P) were prepared by manual mixture of the two proteins in a 1:1 weight ratio, then diluted to a final concentration of 0.5 mg ml?1. Samples (2.7 μl) were applied to a glow-discharged Quantifoil R 2/2 gold grids and vitrified using a Vitrobot Mark IV with a blot time of 3 s before the grid was plunged into liquid ethane. Data were acquired using the Leginon system installed on Titan Krios electron microscopes operating at 300 kV and equipped with a K3-BioQuantum direct detection device. The dose was fractionated over 40 raw frames and collected over a 2-s exposure time. Motion correction, CTF estimation, particle picking, 2D classifications, ab initio model generation, heterogeneous refinements, 3D variability analysis and homogeneous 3D refinements were carried out with cryoSPARC. Local refinement was performed to resolve the RBD–nanobody interface by using a mask encompassing one copy of the RBD–nanobody complex for refinement, after removing the rest of the density by particle subtraction.

  For initial fits to the cryo-EM reconstructed maps, we used the coordinates of the SARS-CoV-2 spike from Protein Data Bank (PDB) code 7JZL, and a nanobody model predicted by the ABodyBuilder server44. These initial models were docked into the cryo-EM maps using Chimera. The coordinates were then fit to the electron density more precisely through an iterative process of manual fitting using Coot and real space refinement within Phenix, Molprobity and EMRinger were used to check geometry and evaluate structures at each iteration step. Figures were generated in UCSF ChimeraX and PyMOL (https://pymol.org). Map-fitting cross correlations were calculated using Fit-in-Map feature in UCSF Chimera. Overall and local resolution of cryo-EM maps was determined using cryoSPARC.

  Sequence entropy are based on nine strains with the following UniProt identifiers: SARS-CoV-2: P0DTD1, B.1.1.7 and B.1.351; SARS-CoV: A7J8L4, Q202E5 and P59594; and bat SARS-like coronavirus: MG772933, WIV16 (A0A0U2IWM2) and RsSHC014 (U5WLK5). The entropy was calculated for each residue based on aligned sequences with the formula:

  $${\rm{Entropy}}\,=-\mathop{\sum }\limits_{i=1}^{21}p(xi)\log (p(xi))$$

  In which xi are standard amino acids, plus gap.

  The buried surface area on the RBD were calculated for 51 human antibody–SARS-CoV-2 RBD complexes using the Naccess program.

  Figures arranged in Adobe Illustrator 2020.

  Further information on research design is available in the?Nature Research Reporting Summary linked to this paper.

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