key: cord-0945501-4zunfua0
authors: Mäkelä, Anna R.; Uğurlu, Hasan; Hannula, Liina; Salminen, Petja; Kant, Ravi; Fagerlund, Riku; Haveri, Anu; Strandin, Tomas; Kareinen, Lauri; Hepojoki, Jussi; Levanov, Lev; Pasternack, Arja; Naves, Rauno A.; Ritvos, Olli; Österlund, Pamela; Sironen, Tarja; Vapalahti, Olli; Kipar, Anja; Huiskonen, Juha T.; Rissanen, Ilona; Saksela, Kalle
title: Intranasal inhibitor blocks Omicron and other variants of SARS-CoV-2
date: 2021-12-28
journal: bioRxiv
DOI: 10.1101/2021.12.28.474326
sha: 48f4b6cf2bd4d299dad03bf17f01fabe354de034
doc_id: 945501
cord_uid: 4zunfua0

The emergence of the SARS-CoV-2 Omicron variant capable of escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung1. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models2-5. However, there remains a need for SARS-CoV-2 blocking agents that are more economical to produce in large scale, while less vulnerable to mutational variation in the neutralization epitopes of the viral Spike glycoprotein. Here we describe TriSb92, a highly manufacturable trimeric human nephrocystin SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of the Spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Delta and Omicron. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before the challenge with SARS-CoV-2 B.1.351 efficiently protected mice from infection. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the Spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. Our results highlight the potential of intranasal inhibitors in protecting susceptible individuals from SARS-CoV-2 infection, and describe a novel type of inhibitor that could be of use in addressing the challenge posed by the Omicron variant.

The success of the current vaccines in the fight against the SARS-CoV-2 pandemic is challenged by the emergence of novel viral variants of concern (VOCs) including Omicron that show resistance to neutralizing antibodies, the faster than expected waning of vaccine-induced protective immunity, and the prevalent vaccine hesitancy in the population. Moreover, immune disorders or other health conditions can preclude appropriate vaccine responses in many individuals. In addition to pharmaceuticals for treating COVID-19 disease, new approaches for preventing transmission and spreading of SARS-CoV-2 are therefore urgently needed.

Since the nasal epithelium of the respiratory tract is the first dominant replication site of SARS-CoV-2 preceding virus transport into the lung 1 , intranasal administration of SARS-CoV-2 neutralizing agents poses an attractive prophylactic concept that could profoundly impact the course and severity of the disease. In animal models, monoclonal antibodies targeted against the Spike envelope protein of SARS-CoV-2 have shown to be effective for COVID-19 prophylaxis 6, 7 . To avoid mutational escape from neutralization, such antibodies have been used as cocktails and targeted against conserved regions of Spike 8 . Markedly increased potency could also be achieved by constructing a pentameric IgM version of Spike-targeted IgG antibodies 4 .

To develop more economical and manufacturable anti-SARS-CoV-2 reagents, many laboratories have moved from monoclonal antibodies to smaller antibody fragments, including single variable domain-based nanobodies. Several of these have shown promising antiviral efficacy in cell culture or animal models, including utility as an intranasal SARS-CoV-2 prophylaxis 9-12 .

Compared to these, small antibody-mimetic scaffold proteins are even more robust, versatile to engineer, and cheaper to produce for diverse biological targeting purposes 13 . Sherpabodies (SH3 Recombinant Protein Affinity) are very small (< 60 aa) targeting modules that constitute a novel addition to the toolbox of scaffold targeting proteins and comprise of an improved SH3-domain fold derived from the human ciliary adaptor protein nephrocystin 14 . Here, we report engineering of a Sherpabody targeted against a highly conserved region in the Spike receptor binding domain (RBD) into a potent prophylactic anti-SARS-CoV-2 agent.

With the aim of developing a novel class of inhibitory proteins that could be used for neutralizing SARS-CoV-2, we screened a large antibody-mimetic phage library displaying ~10 11 unique Sherpabodies ( Fig. 1A and B) by using the receptor binding domain (RBD) of the Spike glycoprotein of the Wuhan-Hu-1 strain as an affinity bait. Starting from the second round of affinity selection and phage amplification (panning), a clear enrichment of RBD-binding clones was observed. After three panning cycles, 192 individual Sherpabody-displaying phage clones were isolated and found to represent 15 different sequences. These 15 unique Sherpabodies were tested in phage-ELISA for binding to RBD-mFc, control mFc, or a monoclonal antibody against the E-tag peptide used for monitoring Sherpabody display efficiency (see Fig. 1B ). All 15 Sherpabody clones mediated strong and apparently specific RBD binding (Fig. 1C ). Clone number 92 (Sb92) was among the strongest RBD binders and was chosen for further development due to its ability to also bind to RBD of SARS-CoV-1 (see later), suggesting a conserved target epitope that would be unlikely to vary between different variants of SARS-CoV-2. Sb92 was produced as a GST-fusion protein in bacteria and its RBD-binding affinity was evaluated using a semi-quantitative antigen capture-ELISA (Fig. 1D) , which indicated an affinity (KD) of 30 nM. Six and three residues in the RT-and n-Srcloops, respectively, of the human nephrocystin SH3 domain were replaced with hexapeptides (in red) comprising random combinations of the 20 natural amino acids to create a large semisynthetic M13 phage library displaying ~10 11 individual Sherpabodies with unique binding surfaces formed by the 12 randomized loop residues shown red in the illustration based on the structure pdb 1S1N 14 . (C) Heat map showing relative binding in an ELISA assay of fifteen affinity-selected Sherpabody-displaying phage clones targeted to RBD of SARS-CoV-2 Spike protein (RBD-mFc), negative control (mFc), or an antibody evenly recognizing all displayed clones (anti-E-tag). (D) Semi-quantitative ELISA-based analysis of the RBD-binding affinity (30 nM) of the Sherpabody clone Sb92.

The potency of Sb92 as a neutralizing agent was tested in a pseudovirus model based on luciferase expressing lentiviral vectors infecting ACE2-expressing HEK293T cells in a Spike-dependent manner 15 . Pseudoviruses carrying Spike proteins corresponding either to the original Wuhan-Hu-1 virus or the B.1.351 (Beta) VOC were incubated with serially diluted concentrations of Sb92 before the infection. Sb92 showed a robust neutralizing capacity with encouraging half-maximal inhibitory concentrations (IC50) of 7 nM and 28 nM against Beta VOC and Wuhan-Hu-1-based pseudoviruses, respectively ( Fig. 2A) .

To enhance the potency and utility of Sb92 as a SARS-CoV-2 inhibitor, we constructed a multimerized derivative of Sb92 (TriSb92) containing three tandem copies of Sb92 connected by flexible 15-mer Gly-Ser, (GGGGS)3, linkers. Remarkably, the trimeric architecture of TriSb92 provided it with an almost 200-fold increased potency to neutralize these pseudoviruses, resulting in IC50 values of 150 pM (Wuhan-Hu-1) and 50 pM (Beta VOC) (Fig. 2B) . Importantly, also the currently most relevant VOCs Delta and Omicron were potently neutralized by TriSb92 and showed IC50 values of 35 pM and 180 pM, respectively (Fig. 2B ). Of note, a pseudovirus carrying the Spike glycoprotein of SARS-CoV-1 could also be readily neutralized by TriSb92, albeit with a somewhat lower efficiency (IC50 6 nM), resembling inhibition of SARS-CoV-2 Spike-mediated infection by the monomeric Sb92 protein (Fig. 2C) .

Subsequently, TriSb92 was tested in a classic coronavirus microneutralization test 16 Together, these data show that TriSb92 is an extremely potent inhibitor of SARS-CoV-2 infection, which targets a conserved site in the RBD of Spike in a manner that is insensitive to immune escape mutations found in the relevant VOCs, including Omicron, and provides TriSb92 with the capacity to neutralize a related sarbecovirus (SARS-CoV-1).

To evaluate the prophylactic efficiency of TriSb92 in vivo, we used a recently described animal This amount of TriSb92 was chosen to represent a relatively modest dose, which is in the range also referred as ultra-low in the SARS-CoV-2 antibody inhibitor literature 2 .

Animals were euthanized at 2 days post infection (dpi) and the right lungs subjected to quantitative real-time PCR (RT-qPCR) for viral RNA. In untreated control mice, the lungs exhibited abundant SARS-CoV-2 subgenomic E RNA (median Cq value 18.45), whereas none could be detected in the lungs of the mice that had received a prophylactic dose of TriSb92. These data are shown in Fig. 3A where mouse actin mRNA-normalized sgE RNA quantitation data are plotted as percentage of the average sgRNA in the five unprotected control mice. The results were confirmed by histology and immunohistology for the detection of SARS-CoV-2 nucleoprotein (NP) in nasal mucosa, airways and left lungs (Fig. 3B ). In control animals, viral antigen was present throughout the respiratory tract, in the nasal cavity (respiratory and olfactory epithelial cells), in bronchiolar epithelial cells and in pneumocytes in adjacent alveoli in association with a mild acute bronchointerstitial pneumonia. Viral antigen was also detected within individual macrophages in bronchiolar lymph nodes. In contrast, treated animals were entirely free of viral antigen in nose, lower airways, lungs and bronchiolar lymph nodes and did not exhibit any pathological changes ( Fig. 3B ). In a second larger experiment, the protective effect of the previously used dose of TriSb92 was compared with a 10-fold lower dose (2.5 µg per nostril) similarly administered 1 h before SARS-CoV-2 challenge. In addition, we tested the effect of the 25 µg per nostril administered 4 h or 8 h before virus inoculation. As shown in Fig. 3 , also the lower dose of TriSb92 efficiently protected the animals from subsequent SARS-CoV-2 challenge 1 h later (no detectable sgE RNA in lungs 3 days postinfection); however, in two of these animals viral antigen was detected in a few patches of bronchiolar epithelial cells. Remarkably, TriSb92 was very effective also when given 4 h before the viral challenge. None of these animals showed evidence of viral antigen expression in the lung, and in only one of the four animals low but detectable levels of sgE RNA were found. Likewise, when TriSb92 was given 8 h before the challenge the lungs of 3 of the 4 mice were clean of sgE RNA and viral antigen, whereas one animal did show substantial albeit reduced viral RNA levels and also expressed viral antigen within bronchiolar epithelial cells.

Together, these data show that intranasal administration of TriSb92 at relatively low doses can provide an impressive prophylactic protection against SARS-CoV-2 infection in this animal model.

To gain structural and mechanistic insight into the broadly cross-neutralizing action of TriSb92, cryogenic electron microscopy (cryoEM) studies were carried out. Single-particle cryoEM analysis of the prefusion SARS-CoV-2 S trimer mixed with TriSb92 resulted in two reconstructions with average estimated resolutions of 3.5 Å and 4.1 Å (Supplementary Fig. 1 , Supplementary Table 1) that display the distinct "up" and "down" conformations characteristic of sarbecoviral RBDs 18-20 .

The first reconstruction, A, describes the S-trimer with all three RBDs in the up-conformation decorated with additional density corresponding to bound TriSb92 ( Fig. 4A and Supplementary Fig.   2 ). The second reconstruction, B, describes the S-trimer with all three RBDs in the downconformation without any additional density indicative of a Sherpabody ( Supplementary Fig. 2 ). To confirm that the additional density associated with the up-conformation RBDs was TriSb92, we performed a fitting of the SH3 domain of human nephrocystin (PDB 1S1N) 14 into the additional density, and found that this density perfectly matched with the size and shape of this SH3 backbone of TriSb92 ( Supplementary Fig. 3 ).

As illustrated in Figs. 4B-D, TriSb92 targets a highly conserved region distal from the ACE2 binding site 21 . In line with the observed cross-neutralization (Fig. 2) , only a single residue within the predicted binding site of TriSb92 diverges between SARS-CoV-2 and SARS-CoV-1, whereas apart from just one of the 15 RBD amino acids changes in Omicron (S375F located at the very edge of the TriSb92 epitope) the mutations found in SARS-CoV-2 VOCs do not involve this binding surface ( Fig. 4C-D) . The epitope of TriSb92 is distinct from the epitopes of therapeutic monoclonal antibodies ( Supplementary Fig. 4 ) that primarily target the ACE2 binding site and remain vulnerable to amino acid changes accruing in this region. Instead, the epitope of TriSb92 partially overlaps with the 'cryptic epitope' targeted by monoclonal antibody (mAb) CR3022 21 ( Supplementary Fig. 4 ) that does not complete with binding to ACE2. Indeed, the ability of ACE2 and Sb92 to simultaneously bind to the RBD in a non-competitive manner could also be experimentally verified using a sandwich-ELISA approach ( Supplementary Fig. 5 ). The binding sites of TriSb92 and ACE2 are highlighted in orange and in purple, respectively. Circles above the RBD residues indicate amino acid changes in Omicron, Delta, Gamma and/or Beta variants, and are coloured as follows: red is shared by Omicron, Gamma, and Beta; yellow is shared by Omicron and Delta; white is unique to Delta; lime green is unique to Omicron.

The emergence of new SARS-CoV-2 variants that compromise vaccine efficacy and are resistant to many clinical therapeutic monoclonal antibodies [22] [23] [24] [25] [26] [27] underscores the importance to develop novel molecular tools that potently neutralize VOCs. The urgency of this notion has been strongly emphasized by the recent rise of the potently immune-evasive and fast-spreading Omicron variant.

In this study, we describe a promising SARS-CoV-2 inhibitor, TriSb92, based on a new and extremely small antibody-mimetic targeting scaffold termed the Sherpabody. TriSb92 targets a highly conserved region and can neutralize all currently known VOCs, including Omicron, and even a related sarbecovirus SARS-CoV-1. This suggests that TriSb92 could be effective against the future immune escape variants of SARS-CoV-2, and potentially also against emerging novel pandemic sarbecoviruses.

An advantage of Sherpabodies over conventional antibodies and most antibody fragments is that they can be readily assembled into multimers in a context-independent manner to enhance avidity, specificity, as well as functionality. Besides the strong avidity effect, the robust SARS-CoV-2 neutralizing potency of TriSb92 appears to stem from its mode of action revealed by our cryoEM studies. Instead of competing with the high-affinity interaction between ACE2 and the RBD of Spike, TriSb92 appears to prevent SARS-CoV-2 entry by triggering an "all three RBDs up" conformation in the Spike trimer. This could destabilize the prefusion spike in a native setting, potentially leading to the spike shedding of the S1 subunit, as has been demonstrated with certain rare antibodies that also target this region in the RBD 28, 29 . Interestingly, the binding site of TriSb92 is inaccessible in the down-conformation of the RBD. We postulate that once one subunit of TriSb92 can bind to a Spike trimer in a "one RBD up" conformation the remaining two free subunits will promote a conformational shift of the two other RBDs until an all-up conformation is achieved. This model is consistent with our findings, that following TriSb92 treatment the S-trimer presents exclusively in all-up or all-down states.

TriSb92 was built from a Sherpabody clone (Sb92) derived from phage-library screening without any further affinity optimization. Thus, it should be relatively facile to achieve an even higher inhibitory potency by affinity maturation of Sb92 and perhaps also by optimization of the trimerization linkers in TriSb92. However, this may not be of great practical relevance considering the already low IC50 concentrations of TriSb92 and the ease of producing it in very high quantities in low-cost bacterial expression systems.

The intranasal dose of TriSb92 that should be administered to susceptible persons prior to events involving a risk for SARS-CoV-2 exposure remains to be established but is likely to be substantially lower than extrapolation of our current data on mice that were challenged by inoculation of the rather massive amount of 2x10 5 PFU of SARS-CoV-2 into their respiratory tract.

Our current data provide a valuable addition to the handful of previous reports on antibody- 

Sherpabody phage display library (size ~10 11 cfu) was obtained from Next Biomed Therapies Oy.

To develop Sherpabodies specific for SARS-CoV-2 spike receptor binding domain (RBD), phage affinity selection process was conducted using standard solid phase sorting strategy. Specific phagedisplayed Sherpabodies were selected for by panning against RBD-mouse IgG2a Fc-fusion protein 32 . Three sequential rounds of off-target depletion and specific panning were performed using a control mFc and specific RBD-mFc, respectively. The immobilized control and target proteins (30 µg/ml in PBS; Maxisorp Immunotubes, Nunc) were sequentially incubated in the presence of infectious naïve Sherpabody phage library in 2.5% milk-PBS-0.1%Tween20, for 2 h (RT) and o/n (4°C), respectively. Non-specific phages were removed by extensive washing (PBS-0.05% PBS-Tween), and the remaining pool of phage were eluted and amplified in E.coli XL1-Blue host cells according to standard protocols. The amplified pool of phages was collected, and the process was reiterated over three rounds to enrich phage-displayed Sherpabodies specific to the RBD-target protein.

Representative transformants from the second and third panning rounds were tested for specific binding to the RBD-mFc target protein by enzyme-linked immunosorbent assay (ELISA).

Mimicking the affinity selection process, in phage-ELISA the target and control proteins were immobilized on an immunoplate (Maxisorp, Nunc). Each single colony represents progeny from a single E. coli cell that harbours a phagemid expressing a unique Sherpabody-pIII fusion protein.

Specifcally, phage-ELISA was performed in 96-well Maxisorp microtiter plates (Nunc) coated over night at 4°C with 100 µl of target and control proteins (10 µg/ml in PBS). The wells were washed 3

x with PBS-0.05% Tween20 and blocked with 5% skimmed milk powder in PBS (milk-PBS) for 2 h at RT. Appropriate dilutions of Sherpabody-displaying single phage clones were prepared in milk-PBS and incubated with the coated target protein for 1 h at RT followed by washes 3 x with PBS-0.05% Tween20 to remove unbound phage. The detection was performed with HRPconjugated mouse monoclonal anti-M13 antibody (GE Healthcare), and TMB (3,3' 5,5'tetramethylbenzidine) substrate. The staining reaction was stopped with 1 M sulfuric acid and absorbance measured at 450 nm using Hidex Sense Microplate Reader.

The DNA encapsulated by the positive phage clones was then sequenced and translated to determine the sequence of the displayed Sherpabody.

The discovered unique Sherpabodies were cloned and expressed in E. coli as monomeric and/or trimeric GST-fusion proteins using standard protocols (GE Healthcare). 15 mer Gly-Ser, (GGGGS)3, linkers were applied in between each fusion partner. GST-tagged Sherpabodies were purified by affinity chromatography using glutathione sepharose according to manufacturer's instructions (GE Healthcare). The buffer was exchanged to PBS by dialysis (140 mM NaCl, 10 mM phosphate buffer, 3 mM KCl, pH 7.4), and the purified Sherpabodies were stored at -80°C and/or at +4°C.

Affinity analysis by semi-quantitative sandwich-ELISA was performed using plastic-coated (o/n, 4°C) GST-Sherpabody fusion protein followed by binding of a concentration series of soluble RBD-His target protein 32 (1 h, RT). The washes and detection was performed as described above using monoclonal HRP-conjugated anti-His antibody.

GST-Sb92/RBD-His/ACE2-mFc sandwich-ELISAs were performed in two ways: first using plastic-coated GST-Sb92 as the capturing reagent (10 µg/ml) and ACE2-mIgG2a as the detection reagent (10 µg/ml) and then vice-versa (10 µg/ml and 5 µg/ml, respectively) to analyse their ability to simultaneously bind to RBD-His 32 (2-fold dilution series starting from 10 µg/ml). The complex formation was detected using either HRP-conjugated goat anti-Mouse-IgG (Sigma Aldrich) or mouse monoclonal anti-GST (GE Healthcare) antibodies. Her2-binding GST-Sb1206 served as a negative control.

HEK293T and HEK293T-ACE2 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2% L-Glutamine, and 1% penicillin/streptomycin (complete medium). Angiotensinconverting enzyme 2 (ACE2) expressing HEK293T cells were generated by lentivirus-mediated gene transduction. Briefly, pWPI-puro plasmid containing ACE2 cDNA (AB046569.1) was co- 

A cytopathic effect (CPE)-based microneutralization (MNT) was performed at the biosafety level 3 laboratory essentially as previously described 33 . Briefly, TriSb92 in duplicates was 2-fold serially diluted in EMEM supplemented with penicillin, streptomycin and 2% of heat-inactivated fetal bovine serum. Virus was added to obtain 100 x TCID50 per well and the 96-well tissue culture plates were incubated for 1 h at +37°C, 5% CO2. African green monkey kidney epithelial (Vero E6) cells were added, following incubation at +37°C, 5% CO2 for 4 days. Wells were fixed with 30% 

RNA was extracted from the right lung of the mice using Trizol (Thermo Scientific) according to the manufacturer's instructions. Isolated RNA was directly subjected to one-step RT-qPCR analysis using TaqMan fast virus 1-step master mix (Thermo Scientific) and AriaMx instrumentation (Agilent) as described previously for E and subE genes 36 . The RT-qPCR for actin was conducted as previously described. 37 Relative quantification of actin-normalized viral RNA levels was achieved by the comparative Ct method 38 using the average of non-treated animals as reference.

The left lung, remaining thoracic organs and heads were trimmed for histological examination.

Heads were sawn longitudinally in the midline using a diamond saw (Exakt 300; Exakt) and gently decalcified in RDF (Biosystems) for 5 days at room temperature and on a shaker. Tissues were routinely paraffin wax embedded. Consecutive sections (3-5 µm) were prepared from lungs and heads and routinely stained with hematoxylin-eosin (HE) or subjected to immunohistochemistry for the detection of SARS-CoV-2 antigen, as previously described 17 .

CoV-2 S, a gene encoding for the prefusion stabilized S protein ectodomain 19 was produced as synthetic cDNA (GeneArt, Life Technologies). The cDNA template encoded for the residues 14−1208 of the original Wuhan-Hu-1 strain S protein (NCBI Reference Sequence:

YP_009724390.1) with prefusion-stabilizing proline substitutions at residues 986 and 987, an abrogated furin S1/S2 cleavage site with a "GSAS" substitution at residues 682-685, and a C- and 'all RBDs down'. These were further separated by hetero refinement applying C3 symmetry.

After local motion correction, particles in each conformation were further refined with non-uniform refinement applying C3 symmetry. Resulting maps were filtered to local resolution for further analysis. CryoEM data collection and processing statistics are presented in Supplementary Table 1 . Unlike structure 7A29, which presents a "2-up, 1-down" RBD conformation, our reconstruction of the S-trimer displays a "3-up" conformation. In order to account for this difference, an RBD was separated from the spike structure in Coot 43 and fitted into the three RBD densities present on the Strimer reconstruction, taking into account the C3 symmetry of the spike. RBD placement yielded a correlation score of 0.8, completing the fitted S-trimer model ( Supplementary Fig. 2) . Furthermore, additional density not comprised of the S-protein was identified adjacent to each RBD (Figure 4 ).

To confirm whether this density was comprised of Sb92, the cryoEM reconstruction was segmented around the SARS-CoV-2 S-protein using 'color zone' (coloring radius of 3Å) and 'split map' functions in Chimera, followed by fitting the structure of human nephrocystin SH3 domain (PDB 1S1N) into the additional density with a fitmap global search. The top solution placed the SH3 domain into the density adjacent to RBDs with a correlation score of 0.88. Furthermore, the top solution places SH3 loops RT and n-Src, that contain antigen-binging mutations in Sb92 (Figure 1 ), on the RBD interface (Supplementary Figure 3) , and points the termini of the protein towards the other Sb92 modules, which is line with the expected binding mode of TriSb92.

Our second reconstruction B (Supplementary Figure 1) , of a closed SARS-CoV-2 spike, was fitted with SARS-CoV-2 structure (PDB 6ZP0 44 , presenting a "3-down" RBD conformation). with ACE2 receptor and inhibitors, including TriSb92 and nanobodies VHH-V and Nb30, and the ACE2 receptor and inhibitors including TriSb92 and therapeutic monoclonal antibodies were identified using the PDB ePISA server.

The cryo-EM density maps of i) SARS-CoV-2 spike with RBDs in the "up"-conformation and decorated with TriSb92 (accession code EMD-25781) and ii) SARS-CoV-2 spike with RBDs in the "down" conformation (EMD-25782) have been deposited in the EMDB at the EBI. Fitted SARS-CoV-2 spike coordinates have deposited in the PDB (accession codes PDB 7TAQ and 7TAR, for the "up" and "down" conformations, respectively). All data generated or analysed during this study are included in the main text and its supplementary information files. 

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GST-Sb1206 was also tested as a negative control (Control coated). A constant amount of the capturing and detector reagents were used in each case, while increasing concentrations (0 -385 nM) of RBD were added as a bridging reagent to test the formation of a trimeric Sb92-RBD-ACE2 complex, which was visualized using a HRP-conjugated antibody against the detector.

B.

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