key: cord-0428648-yxpja8m4
authors: Maeda, Ryota; Fujita, Junso; Konishi, Yoshinobu; Kazuma, Yasuhiro; Yamazaki, Hiroyuki; Anzai, Itsuki; Yamaguchi, Keishi; Kasai, Kazuki; Nagata, Kayoko; Yamaoka, Yutaro; Miyakawa, Kei; Ryo, Akihide; Shirakawa, Kotaro; Makino, Fumiaki; Matsuura, Yoshiharu; Inoue, Tsuyoshi; Imura, Akihiro; Namba, Keiichi; Takaori-Kondo, Akifumi
title: Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants
date: 2021-10-26
journal: bioRxiv
DOI: 10.1101/2021.10.25.465714
sha: e460897aa9e4723b5109d77352fd4d24048b2c0a
doc_id: 428648
cord_uid: yxpja8m4

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted—most successfully, vaccination1–3—imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)—namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

7 First, we measured the binding kinetics of these clones for the whole extracellular domain 109 of SARS-CoV-2 spike, which was the same construct that was used for immunization, 110 via biolayer interferometry (Fig. 1e) . We noticed that the salt concentrations in the kinetic 111 assay buffers affected the avidities of the clones. Of note, high salt concentrations were 112 necessary to stabilize the spike complex (Extended Data Fig. 1b) . Four clones (P17, P86, 113 C116, and P334) showed high avidity for the SARS-CoV-2 spike complex in hypertonic 114 buffer; the others (C17, C49, P158, C246, and P543) showed high avidity in hypotonic 115 buffer (Extended Data Fig. 2c,d) . 116

Second, we checked these nanobodies via microscopy observation using HEK 117 cells expressing the SARS-CoV-2 spike. Seven clones stained near the cell surface; two 118 clones (C49 and C246) stained only an intracellular region (Extended Data Fig. 3a,b) . 119

Third, flow cytometric analysis supported this observation: the seven clones detected the 120 spike protein on the cell surface, but the C49 and C246 clones did not (Extended Data 121 Approximately 100,000 paired reads of each library were generated. The raw data of 548 reads were trimmed of the adaptor sequence using cutadapt v1.18 59 , and low-quality 549 reads were subsequently removed using Trimmomatic v0.39 60 . The remaining paired 550 reads were merged using fastq-join 61 and then translated to the amino acid sequences 551 using EMBOSS v6.6.0.0 62 . Finally, unique amino acid sequences in each library were 552 counted using a custom Python script combining seqkit v0.10.1 63 and usearch v.11 64 . 553 Enrichment scores of each clone were analysed by calculating the P-value of c 2 tests 554 32 between the existing ratios among the different sublibraries. We chose nine clones 555 whose enrichment scores in the SARS-CoV-2 spike biopanned sublibraries were higher 556 than those in other biopanned sublibraries. 557 558

Each selected amino-acid sequence was connected with a (GGGGS) 4 linker as a tandem 560 dimer; coding genes of these and of the previously reported SARS72 dimer, mNb6 561 dimer, and Ty1 monomer were codon-optimized and synthesized (Eurofins Genomics 562 Inc, Tokyo, Japan). The synthesized genes were subcloned in the pMES4 vector to 563 express N-terminal PelB signal peptide-conjugated and C-terminal 6×His-tagged 564 nanobodies into the bacterial periplasm. These gene constructs were transformed into 565 BL21(DE3) E. coli cells (BioDynamics Laboratory Inc., Tokyo, Japan) and plated on 566 LB agar with ampicillin, which were incubated at 37°C overnight. Colonies were picked 567 and cultured at 37°C to reach an OD of 0.6 AU; the cells were cultured at 37°C for 3 h 568 or at 28°C overnight with 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside: Nacalai). 569 

UCSF Chimera--a visualization system for exploratory 970 research and analysis

Features and development 973 of Coot

PHENIX: a comprehensive Python-based system for 976 macromolecular structure solution

MolProbity: all-atom structure validation for macromolecular 979 crystallography

Structure visualization for researchers

How good are my data and what is the 986 56 resolution?

Molecular replacement with MOLREP

peptide-binding nanobody for proteomics and microscopy

REFMAC5 for the refinement of macromolecular 995 crystal structures

Overview of the CCP4 suite and current developments

Acknowledgments This study was supported by donations from POPURI Pharmacy Co

Japan), grants from the AMED Research Program on Emerging and Re-1003

emerging Infectious Diseases

develop at room temperature for 30 min, after which the reaction was stopped with the 681 addition of 10 µl of 5 M sulfuric acid (H 2 SO 4 ). Each well was read at an optical density 682 (OD) of 450 nm using a microplate reader (Bio-Rad). 683 684 Immunochromatography 685Antigen test kits detecting the SARS-CoV-2 spike based on nitrocellulose lateral flow 686 assays were developed by Yamato Scientific Co., Ltd. (Tokyo, Japan). Briefly, purified 687 P158 (330 ng µl -1 ) was lined approximately 1 mm wide on an IAB90 nitrocellulose 688 membrane (Advantech, Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The membrane was 689 soaked in 1×Carbo-Free blocking solution at room temperature for 1 h and air dried. 690Purified P86 or P543 nanobodies were amine coupled to 30-nm diameter Estapore beads 691 (Sigma-Aldrich) according to the manufacturer's protocol. Glass fibre paper (GF/DVA: 692 Cytiva) was soaked in blocking solution containing 0.005% (w/v) nanobody-coupled 693Estapore beads for saturation and then dried under vacuum conditions. The lined 694 nitrocellulose membrane and the bead-absorbed glass fibre paper were overlaid on a 695 backing sheet (Cytiva). CF4 paper (Cytiva) was used for both the sample pad and the 696 absorbance pad; they were overlaid on the prepared backing sheet as well. The four-697 layered sheet was cut 5 mm wide and housed in black cases: the cassettes were stored in 698 40 sealed packages with silica gels. When dried, 150 µl of sample was spotted onto the 699 sample pad; the kits were photographed under a 315 nm UV lamp for an arbitrary amount with shaking at 1,000 rpm. Biotin-conjugated clones at 10 µg ml -1 were captured on a 709 streptavidin-coated sensor chip (SA: fortèBIO) to reach the signals at 4 nm. One uncoated 710 sensor chip was monitored as the baseline; another biotin-conjugated anti-IL-6-coated 711 sensor chip (anti-IL-6 nanobody: COGNANO Inc.) was monitored as the background. 712The remaining 6 channels were immobilized with biotinylated anti-SARS-CoV-2 spike 713 clones, and real-time binding kinetics to the purified extracellular domain of the SARS-714CoV-2 spike trimer complex were measured in sequentially diluted concentrations at the 715 same time (8 channels per assay). The concentrations of the SARS-CoV-2 spike loaded 716 41 varied between 1-32 µg ml -1 , corresponding to 1-32 nM or less, when an average of the 717 molecular weight of the SARS-CoV-2 spike trimer complex was estimated to be 718 approximately 1,000 kDa or more according to chromatograms of gel filtration column 719 chromatography (Extended Data Fig. 1b The images were processed using RELION 3.1 69 . Movies were motion corrected using 835MotionCor2 70 , and the contrast transfer functions (CTFs) were estimated using CTFFIND 836 4.1 71 . Micrographs whose CTF max resolutions were beyond 5 Å were selected. Three-837 dimensional (3D) template-based autopicking was performed for all images, and the 838 particles were extracted with 4× binning, which were subjected to two rounds of 2D 839classification. An initial model was generated and used as a reference for the following 840