An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

Authored by nature.com and submitted by mvea
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Vero E6 cells (ATCC-CRL-1586) and HEK293T cells (ATCC-CRL-3216) were maintained in Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% fetal calf serum and 1% penicillin–streptomycin and cultured at 37 °C in a humidified incubator with 5% CO 2 . A HEK293T cell line engineered to overexpress human ACE2 (HEK293T-ACE2) was generated by the lentiviral transduction of HEK293T cells. Briefly, lentiviruses were produced by co-transfecting HEK293T cells with a plasmid encoding VSV-G (Addgene cat#12259), a lentiviral Gag-Pol packaging plasmid (Addgene cat#8455), and a human ACE2 transfer plasmid. Virions were harvested from the supernatant, filtered through 0.45 µm filters, and used to transduce HEK293T cells. All cell lines used for experiments were negative for Mycoplasma as determined by PCR. Infectious SARS-CoV-228 was propagated in Vero E6 cells and titrated by plaque assay.

The plasmid for expression of the SARS-CoV-2 prefusion-stabilized spike ectodomain with a C-terminal T4 fibritin trimerization motif was obtained from ref. 2. The plasmid was used to transiently transfect FreeStyle 293F cells using FreeStyle MAX reagent (Thermo Fisher Scientific). The S ectodomain was purified from filtered supernatant on Streptactin XT resin (IBA Lifesciences), followed by size-exclusion chromatography on a Superdex 200 in 5 mM Tris pH 8, 200 mM NaCl.

The RBD domain (RVQ-VNF) of SARS-CoV-2 was cloned upstream of an enterokinase cleavage site and a human IgG1 Fc. This plasmid was used to transiently transfect FreeStyle 293F cells using the FreeStyle MAX reagent. The RBD-Fc fusion was purified from filtered supernatant on Protein G Sepharose (GE Healthcare). The protein was cleaved using bovine enterokinase (GenScript) leaving a FLAG-tag at the C-terminus of the RBD. Enzyme and Fc-portion were removed on HIS-Pur Ni-NTA resin (Thermo Fisher Scientific) and Protein G sepharose (GE Healthcare), respectively, and the RBD was purified by size-exclusion chromatography on a Superdex 200 in 50 mM Tris pH 8, 200 mM NaCl.

In addition, the RBD domain (RVQ-VNF) was cloned upstream of a Sortase A recognition site (LPETG) and a 6xHIS tag and expressed in FreeStyle 293F cells as described above. RBD-HIS was purified from filtered supernatant on His-Pur Ni-NTA resin, followed by size-exclusion chromatography on a Superdex 200.

The nanobodies were cloned for expression in the pHEN plasmid with a C-terminal Sortase recognition site (LPETG) and a 6xHIS tag. This plasmid was used to transform BL21 cells for periplasmic expression. Expression was induced with 1 mM IPTG at OD600 = 0.6; cells were grown overnight at 30 °C. Nanobodies were retrieved from the periplasm by osmotic shock and purified by Ni-NTA affinity purification and size-exclusion chromatography.

Biotinylated and fluorescent probes were generated using Sortase A as described in refs. 29,30. In brief, nanobodies were site-specifically biotinylated on the C-terminus using Sortase A 5 M. Nanobody at a concentration of 50 μM was incubated with sortase A 5 M (5 μM), GGGK-biotin (200 μM) in 50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM CaCl 2 , for 2 h at 25 °C. Unreacted nanobody and sortase was removed with Ni-NTA resin and excess GGGK-biotin was removed using Zeba spin desalting columns (0.5 ml, 7k MWCO, Thermo Fisher Scientific).

To generate the fluorescently labeled probes, first a dibenzocyclooctyne-amine (DBCO-amine, Sigma Aldrich) was attached via sortase A to the nanobody or the RBD (reaction conditions: 50 μM RBD or nanobody, 50 μM Sortase A 5 M, 8 mM DBCO-amine in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl 2 , 2 h, 25 °C). Unreacted probe, sortase and excess DBCO-amine were removed using Ni-NTA resin and PD-10 columns (GE Healthcare), respectively. Abberior Star 635P-azide (Abberior GMBH) was attached to the DBCO-labeled proteins in a copper-free click chemistry reaction. Unreacted fluorophore was removed on PD-10 column (RBD) or size-exclusion chromatography (nanobody).

For mammalian expression, the sequence encoding the nanobody Ty1 was cloned upstream of a human IgG1. This plasmid was used to transiently transfect FreeStyle 293F cells using the FreeStyle MAX reagent. The Ty1-Fc fusion was purified from filtered supernatant on Protein G Sepharose followed by size-exclusion chromatography.

Alpaca immunization and phage display was performed similarly as described in refs. 31,32. In brief, the adult male alpaca Tyson at PreClinics, Germany, was immunized four times in a 60-day immunization schedule. SARS-CoV-2 S1-sheep-Fc (Native Antigen Company, SKU: REC31806) was used for the first two immunizations, and SARS-CoV-2 RBD produced in FreeStyle 293F cells was used for the last two immunizations. The animal study protocol was approved by the PreClinics animal welfare officer commissioner and registered under the registration No. 33.19-42502-05-17A210 at the Lower Saxony State Office for Consumer Protection and Food Safety—LAVES and is compliant with the Directive 2010/63/EU on animal welfare.

Four days after the final boost, RNA was isolated from PBMCs (RNA Plus mini kit, Qiagen). For cDNA synthesis, SuperScript III RT (Thermo Fisher Scientific) was used with a combination of oligo(dT), random hexamers, or gene-specific primers (AL.CH2, ATGGAGAGGACGTCCTTGGGT, and AL.CH2.2 TTCGGGGGGAAGAYRAAGAC)32. All primer sequences are listed in Supplementary Table 2. Nanobody sequences were PCR amplified and cloned into a phagemid vector for expression as pIII fusion. TG1 cells (Lucigen) were transformed with this library by electroporation.

Cells were inoculated with VCSM13 helper phage, and the resulting phage was enriched in two consecutive rounds of phage display on RBD immobilized on magnetic beads. After the second round of phage display, individual bacterial colonies were picked in a 96-well format, grown until OD = 0.6 and nanobody expression was induced by addition of 1 mM IPTG. After 16 h incubation at 30 °C, bacterial supernatant was used as primary detection reagent in an ELISA coated with RBD or S ectodomain. Bound nanobodies were detected with anti-E tag (Bethyl laboratories, 1:10,000) secondary antibody. Positive clones were sequenced and cloned into the pHEN expression vector for further characterization.

NGS and analysis of nanobody libraries

Plasmids from nanobody libraries before enrichment, and after each enrichment step, were amplified for 13 cycles using Q5 High-Fidelity 2X Master Mix (NEB) according to manufacturer’s instructions, using primers: NB-NGS-FW: CACTCTTTCCCTACACGACGCTCTTCCGATCTCTCGCGGCCCAGCCGGCCATGG and NB-NGS-RV: GGAGTTCAGACGTGTGCTCTTCCGATCTACCGGCGCACCACTAGTGCA, annealing at 72 °C. Illumina indexing primers were added using an additional 9 cycles, with Kapa HiFi. Amplicons were size selected using Agencourt AMPure XP beads (bead ratio: 1:1), and were pooled at ratios of 6:2:1 for pre:post-1:post-2 libraries, to account for the reduction in diversity expected during enrichment, and sequenced on an Illumina MiSeq using the MiSeq Reagent Kit v3 (2 × 300) MS-102-3003.

Paired-end reads were merged using USEARCH1133, and then processed in the Julia language, primarily using the NextGenSeqUtils.jl package34 (analysis code is available here: https://github.com/MurrellGroup/Ty1). Briefly, reads are trimmed of primer sequences, and deduplicated, maintaining read frequencies. Variant frequencies are calculated as combined frequency of any reads matching a variant within 3% nucleotide divergence, using a kmer-based distance approximation for rapid database search. Any reads with counts >3 from the second enrichment library are searched for their variant frequencies across all databases. When calculating enrichment, to avoid zeros due to sampling and to regularize against over-sensitivity to low-frequency baseline variants, all frequencies are increased by the reciprocal of the size of the pre-enrichment database.

Pseudotyped viruses were generated by the co-transfection of HEK293T cells with plasmids encoding the SARS-CoV-2 spike protein harboring an 18 amino acid truncation of the cytoplasmic tail35, a plasmid encoding firefly luciferase, and a lentiviral packaging plasmid (Addgene cat#8455) using Lipofectamine 3000 (Invitrogen). Media was changed 12–16 h after transfection, and pseudotyped viruses were harvested at 48 and 72 h post transfection, filtered through a 0.45 µm filter, and stored at −80 °C until use. Pseudotyped virus neutralization assays were adapted from protocols previously validated to characterize the neutralization of HIV36, but with the use of HEK293T-ACE2 cells. Briefly, pseudotyped viruses sufficient to generate ~100,000 RLUs were incubated with serial dilutions of nanobodies for 60 min at 37 °C. Approximately 15,000 HEK293T-ACE2 cells were then added to each well and the plates were incubated at 37 °C for 48 h. Luminescence was then measured using Bright-Glo (Promega) per the manufacturer’s instructions on a GM-2000 luminometer (Promega) with an integration time of 0.3 s.

Cells were trypsinized and fixed in 4% formaldehyde/PBS and stained with RBD-AS635P under non-permeabilizing conditions or with Ty1-AS635P under permeabilizing conditions. Fluorescence was quantified using a BD FACSCelesta and the FlowJo software package.

Vero E6 cells were seeded onto coverslips in a 24-well plate and incubated overnight at 37 °C/5% CO 2 . Cells were infected with SARS-CoV-2 at a MOI of 1 for 24 h. Cells were fixed with 4% (v/v) formaldehyde, permeabilized in 0.5% Triton X-100 and blocked in 5% horse serum. Cells were incubated with anti-dsRNA antibody (1:2000, J2 Scicons, RNT-SCI-10010200) for 1 h at room temperature followed by 1 h staining with the secondary antibody anti-mouse-Alexa Fluor 488 (1:2000, Thermo Fisher Scientific, A-21202), Hoechst (1:1000, Invitrogen) and Ty1-AS635P (0.5 µg/ml). Coverslips were mounted in mounting media and images were obtained using Zeiss Axiovert microscope and processed using Adobe Photoshop.

BLI was performed using single-use high-precision streptavidin biosensors (SAX) on an eight-channel Octet RED instrument according to manufacturer’s protocols (Fortebio)16. Assays were performed in 2xPBS comprising 0.05% Tween-20 (PBST). Biotinylated nanobodies Ty1 and NP-VHH1 were loaded at concentrations between 30 and 250 nM followed by quenching using biocytin to reach final sensor loads of between 0.15 and 0.7 nm. For the comparative binding test, the eight sensors were divided into two sets, each comprising double sample as well as single reference and single control sensors. Sample and reference sensors were loaded with respective nanobodies. The SAX control was only quenched. Loading of the two sets was performed consecutively to reach similar immobilization levels, while subsequent association and dissociation phases were performed simultaneously. For association, the sample and control sensors were dipped into RBD, while the reference sensor was dipped into PBST. For titration experiments, all sensors were loaded simultaneously. During association one of the sensors was used as reference and only dipped into PBST. Raw data were pre-processed, analyzed, and fitted by applying the 1:1 binding model as implemented in the manufacturer’s software. Bayesian analysis to obtain the two-dimensional distribution of K D and k off -rate values were performed using Evilfit12,13,14. The shown titration data were processed applying reference sensor subtraction and Savitzky–Golay filter operations.

For ITC, proteins were exchanged to 2xHBS-buffer (50 mM HEPES, 300 mM NaCl, pH 7.5) and isolated as single peak populations by Superdex-200 HR10/300 size-exclusion chromatography. ITC measurements were performed using an ITC200 calorimeter (GE Healthcare). The cell temperature was set to 37 °C and the syringe stirring speed to 750 rpm. Before each experiment, the RBD and nanobodies were loaded into the cell and syringe at concentrations of 4 and 75 μM, respectively. Data and binding parameters were analyzed using the MicroCal PeakITC software (Malvern). The integrated heat versus molar ratio plots of the Ty1:RBD interactions were obtained by subtracting the Ty1 dilution heat uptake from the binding data. The NP-VHH1:RBD data were only baseline-corrected, since dilution effects were not evident.

Raw and processed BLI/ITC data were imported into Rstudio for visualization and further analysis37,38,39. Data along with analysis R scripts will be made publicly available via Github and/or DataDryad.

Spike trimer (0.7 mg/ml) and Ty1 (1.3 mg/ml) were mixed in a 1:8 molar ratio and incubated on ice for 5 min. A 3-μl aliquot of the sample solution was applied to glow-discharged CryoMatrix holey grids with amorphous alloy film (Zhenjiang Lehua Technology) in a Vitrobot Mk IV (Thermo Fisher Scientific) at 4 °C and 100% humidity (blot 10 s, blot force 3).

Cryo-EM data collection was performed with EPU 2.7 (Thermo Fisher Scientific) using a Krios G3i transmission-electron microscope (Thermo Fisher Scientific) operated at 300 keV in the Karolinska Institutet 3D-EM facility. Images were acquired in nanoprobe EFTEM mode with a slit width of 10 eV using a GIF 967 energy filter (Ametek) and a K3 detector (Ametek) during 2.4 s with a dose rate of 4.1 e−/px/s resulting in a total dose of 38 e−/Å2 fractionated into 40 movie frames. Motion correction, CTF-estimation, Fourier binning (to 1.02 Å/px), picking and extraction in 428 pixel boxes were performed on the fly using Warp40. A total of 13,589 micrographs were selected based on an estimated resolution cutoff of 4 Å and defocus below 2 microns and 573,036 particles were picked by Warp. Extracted particles were imported into cryoSPARC v2.15.041 for 2D classification, 3D classification, and non-uniform 3D refinement. The particles were processed with C1 symmetry throughout. After 2D classification (300 classes) 354,678 particles were retained and used to build three ab-initio 3D reconstructions. These were further processed for heterogeneous refinement that resulted in one reconstruction showing high-resolution structural features in the core of the spike. One round of homogenous refinement followed by non-uniform refinement resulted in a final reconstruction to an overall resolution of 2.9 Å (0.143 FSC) using 210,832 particles. Localized reconstruction42 were performed using particles where all parts of the spike except the N-terminal domains, the RBDs, and the nanobodies had been subtracted43. The combined effects of these two approaches significantly increased the level of density detail in the upper part of the spike.

A structure of the 2019-nCoV spike protein trimer2 (PDB: 6VSB) was used as a starting model for model building. The model was extended and manually adjusted in COOT44. The nanobody structure was homology modeled using SWISS-MODEL45 taking PDB:5JMR46 as a template. The missing regions of the RBD domains were built based on the RBD-ACE2 crystal structure (PDB: 6LZG)47. For model building and refinements, a composite map was made using PHENIX48 utilizing the particle center-of-mass focused reconstruction and the map from the localized reconstruction described above.

Structure refinement and manual model building were performed using COOT and PHENIX in interspersed cycles with secondary structure and geometry restrained. All structure figures and all EM density-map figures were generated with UCSF ChimeraX49.

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

Sash0 on October 5th, 2020 at 00:58 UTC »

How is the nanobody supposed to be administered? How would our immune system react to it?

baselq1996 on October 4th, 2020 at 23:57 UTC »

I don't understand how pharmaceutical stuff work, do they just randomly go try stuff from alpaca or a kangaroo and see what happens? or alpacas have been used before in this field?

mvea on October 4th, 2020 at 22:31 UTC »

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

Hanke, L., Vidakovics Perez, L., Sheward, D.J. et al.

Nature Communications volume 11, Article number: 4420 (2020)

DOI: https://doi.org/10.1038/s41467-020-18174-5

Abstract

SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.