key: cord-0721475-rwc1sbx4
authors: Low, Jun Siong; Jerak, Josipa; Tortorici, M. Alejandra; McCallum, Matthew; Pinto, Dora; Cassotta, Antonino; Foglierini, Mathilde; Mele, Federico; Abdelnabi, Rana; Weynand, Birgit; Noack, Julia; Montiel-Ruiz, Martin; Bianchi, Siro; Benigni, Fabio; Sprugasci, Nicole; Joshi, Anshu; Bowen, John E.; Walls, Alexandra C.; Jarrossay, David; Morone, Diego; Paparoditis, Philipp; Garzoni, Christian; Ferrari, Paolo; Ceschi, Alessandro; Neyts, Johan; Purcell, Lisa A.; Snell, Gyorgy; Corti, Davide; Lanzavecchia, Antonio; Veesler, David; Sallusto, Federica
title: ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies
date: 2022-03-30
journal: bioRxiv
DOI: 10.1101/2022.03.30.486377
sha: e17fb047a92c8fdf24b6d540d95c5b163be7f781
doc_id: 721475
cord_uid: rwc1sbx4

Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing ‘2P’ mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.

Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing '2P' mutations and becomes exposed upon binding of ACE2 or ACE2mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.

Human-infecting coronaviruses (HCoVs) are distributed across two genera: alphacoronavirus, which includes NL63 and 229E, and betacoronavirus, which includes OC43, HKU1, SARS-CoV-2, SARS-CoV and MERS-CoV. The spike (S) glycoprotein, which facilitates viral entry into host cells via ACE2 receptor, is composed of the S1 and S2 subunits, is highly divergent, with only ~30% sequence identity between alpha and beta coronaviruses and is the main target of neutralizing antibodies (1) (2) (3) . Previous studies have described neutralizing monoclonal antibodies (mAbs) that cross-react amongst sarbecoviruses by targeting the receptor binding domain (RBD) (4) (5) (6) (7) (8) (9) (10) or more broadly across beta coronaviruses by targeting the stem helix (11) (12) (13) (14) (15) . However, neutralizing mAbs that target both the alpha and beta coronaviruses have not been reported.

Broadly neutralizing mAbs, as exemplified for those targeting influenza viruses or HIV-1 (16) (17) (18) (19) (20) (21) , can potentially be used for prophylaxis or therapy and to guide the design of vaccines eliciting broadly protective immunity (22, 23) .

To search for rare antibodies that cross-react with alpha and beta coronaviruses, we stimulated under limiting conditions, total peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2 immune donors, in the presence of R848 and IL-2, which selectively induce the proliferation and differentiation of memory B cells (24) . On day 12, the specificities of IgGs secreted in the culture supernatants were tested by ELISA against a panel of recombinant S proteins from beta and alpha HCoVs (Fig. 1, A to C) . The number of SARS-CoV-2 IgG-positive cultures was generally higher in COVID-19 convalescent patients and in SARS-CoV-2 vaccinees with prior infection (preimmune) as compared to vaccinees without prior infection (naïve). Most SARS-CoV-2 IgGpositive cultures were either monospecific or cross-reactive with the closely related SARS-CoV, while a small fraction was also reactive with OC43 and HKU1 (Fig. 1, A to D) , consistent with previous serological analyses (25) (26) (27) . Remarkably, six cultures (out of >4,000) from 5 individuals (out of 43) cross-reacted with all alpha and beta HCoV S proteins tested (Fig. 1, A to D) , suggesting that memory B cells producing pan-coronavirus antibodies might exist at very low frequency.

To isolate pan-coronavirus mAbs, we combined the broad screening of polyclonallyactivated memory B cells with the sorting and cloning of antibody secreting cells to retrieve paired heavy and light chain sequences (Fig. S1) . Using this approach, we isolated 16 SARS-CoV-2-S-specific mAbs that cross-reacted with various HCoV S proteins (Fig. 1E and Fig. S2A ). Six mAbs (Group 1) cross-reacted with SARS-CoV, OC43 and HKU1 S proteins and bound with EC50 values ranging from 45 ng/ml to 3,000 ng/ml. Three mAbs (Group 2) cross-reacted with all beta HCoV S proteins with high-avidity, as illustrated by their EC50 values ranging from 18 ng/ml to 40 ng/ml (Fig. 1E) . These mAbs were found to target the stem helix region and were described in a separate study (11) . Remarkably, the remaining seven mAbs (Group 3) exhibited the broadest cross-reactivity to both alpha and beta HCoVs with EC50 values ranging from 29 ng/ml to 800 ng/ml (Fig. 1E) . These pan-reactive mAbs, which will be the focus of the present study, were isolated from convalescent or vaccinated individuals, used different V genes (except for C13B8 and C13A7 that were clonally related) and displayed a high load of somatic mutations (7-14% in VH, 2-8% in VL at the nucleotide level, Table S1 ). These results illustrate the utility of a simple high-throughput method based on multiple parallel screening steps of memory B cells to isolate broadly reactive and even pan-reactive coronavirus mAbs.

Using SARS-CoV-2 S1, RBD and S2 proteins, as well as 15-mer linear peptides covering the entire S sequence, the specificities of all seven pan-coronavirus mAbs were mapped to the K811PSKRSFIEDLLFNK825 sequence in the S2 subunit ( Fig. 2A) . This sequence spans the S2' cleavage site (R815) and the fusion peptide N-terminal region, which is essential for membrane fusion (28) and is highly conserved amongst all genera of the Orthocoronavirinae subfamily, including the alpha, beta, gamma, and delta coronaviruses, as well as all SARS-CoV-2 variants sequenced to date (Fig. S3, A and B) . Notably, the seven pan-coronavirus mAbs bound with different EC50 to the prefusion HexaPro and PentaPro S trimers as well as to prefusion and postfusion S2, likely due to the presence of the F817P mutation in HexaPro S ( Fig. 2A) .

To explore the ontogeny of fusion peptide-specific pan-coronavirus mAbs, we compared the binding properties of mature mAbs to their unmutated common ancestors (UCAs) (Fig. 2B and   Fig. S2B ). The UCAs of C13B8, C13A7 (the two clonally related mAbs) and VN01H1 already exhibited broad reactivity to HCoV S proteins but had lower affinity. In contrast, the C13C9 UCA bound only to the beta HCoVs OC43 and MERS-CoV S, whereas the VP12E7 UCA bound only to the alpha HCoV NL63 S, with low avidity in both cases. Finally, the UCAs of C28F8 and C77G12 did not bind to any HCoV S proteins tested. Given the lack of a common V gene usage, these findings suggest that pan-reactive fusion peptide-specific mAbs can mature through multiple pathways and acquire high affinity and cross-reactivity through somatic mutations, possibly as a consequence of priming by endemic coronavirus infection followed by SARS-CoV-2 boost.

Considering the conservation of the fusion peptide region, we next asked if pan-or broad reactivity would be a property shared by most fusion peptide-specific antibodies. IgGs secreted upon polyclonal activation of memory B cells from 71 convalescent individuals were screened for binding to a pool of peptides comprising the fusion peptide sequences of alpha and beta HCoVs, as well as to SARS-CoV-2 S protein. Cultures producing fusion peptide-specific antibodies were detected at low frequency and only in 19 individuals (Fig. S4A) . Although nearly all fusion peptide-reactive antibodies bound to SARS-CoV-2 S protein, only 9 out of 30 were pan-reactive, while the remaining antibodies showed different degrees of cross-reactivity (Fig. S4B) . Thus, although the fusion peptide region in the coronavirus S protein is immunogenic (29) (30) (31) (32) (33) and

conserved, pan-reactivity is the property of a minority of fusion peptide-specific mAbs.

We next tested the neutralizing activity of all seven mAbs against alpha and beta HCoV pseudoviruses in TMPRSS2-expressing target cell lines. Despite binding to the same motif, these mAbs displayed heterogeneous neutralizing potencies. Most notably, VN01H1 and VP12E7 neutralized all pseudotyped alpha and beta HCoVs tested (SARS-CoV-2, SARS-CoV, MERS-CoV, NL63 and 229E), as well as pre-emergent bat sarbecovirus WIV-1 ( Fig. 2C and Fig. S5, A and B). In addition, C77G12 neutralized all beta coronaviruses and showed efficient neutralization of SARS-CoV-2 Wuhan-1 and Omicron BA.1 and inhibition of cell-cell fusion (Fig. 2, D and E) .

These results suggest that fusion peptide-specific mAbs can impede S proteolytic activation or fusogenic rearrangements, thereby inhibiting membrane fusion.

In view of the broad reactivity of VN01H1 and the relatively high potency of C77G12, we further assessed the protective efficacy of these mAbs in vivo. In the Syrian hamster model of SARS-CoV-2 P.1 (Gamma) infection, prophylactic administration of either mAb at high doses reduced RNA copies and lung viral titers, and ameliorated lung pathology, in a statistically significant manner (Fig. 2F) . In summary, these findings demonstrate that fusion peptide-specific mAbs display protective efficacy in vivo, albeit with moderate potency.

To gain insights into the epitope recognized by fusion peptide-specific pan-coronavirus mAbs, we first performed substitution scan analysis on the six clonally unrelated mAbs. All mAbs displayed a core binding motif at I818EDLLFNK825 (Fig. S6A) but C28F8, C77G12, VN01H1, and VP12E7, showed a 3-amino acid expanded footprint spanning the N-terminal R815SF817 residues, comprising the S2' cleavage site at position R815.

We then determined crystal structures of five mAbs (C13B8, C13C9, C77G12, VN01H1 and VP12E7) in complex with the K811PSKRSFIEDLLFNK825 fusion peptide at 2.1 Å, 2.1 Å, 1.7 Å, 1.86 Å and 2.5 Å, resolution, respectively ( Fig. 3 and Fig. S6 , B to D and Table S2 ). All five mAbs bind to overlapping epitopes in the fusion peptide through interactions involving the heavy and light chains. The strict conservation or the conservative substitution of key residues involved in mAb recognition (R815, S816, I818, E819, D820, L821, L822, F823, N824 and K825) across the Orthocoronavirinae subfamily explains the unprecedented cross-reactivity of these fusion peptidespecific mAbs ( Fig. S3 and Table S4 ). The overall architecture of the fusion peptide in the C13C9bound complex structure is most similar to that observed in the prefusion S trimer (PDB 6VXX) (3, 34) (Fig. 3C and Fig. S6B) . The SARS-CoV-2 S fusion peptide adopts a similar conformation in the two structures determined in complex with VN01H1 or VP12E7, which is distinct from the conformation observed in prefusion SARS-CoV-2 S trimeric structures (3, 34) (Fig. 3 , B and C and Fig. S6C ). Specifically, residues 813SKR815 refold from an extended conformation in prefusion S to an ɑ-helical conformation in the two Fab-bound peptide structures, thereby extending the ɑhelix found at the N-terminal region of the fusion peptide. The fusion peptide residues P812-R815 adopt an extended conformation, distinct from prefusion S, in the C77G12-bound complex structure (Fig. 3, A and C) , whereas these residues are disordered in the C13B8-bound structure ( Fig. 3C and Fig. S6D ). The conserved residue R815, which is the S2' site of proteolytic processing upon receptor binding for membrane fusion activation, is engaged in electrostatic interactions with the C13C9, VN01H1, VP12E7 and C77G12 Fabs and therefore buried at the interface with their paratopes (Table S4) . Since pre-incubation of a soluble native-like SARS-CoV-2 S ectodomain trimer with fusion peptide-specific mAbs did not prevent S2X58-induced triggering of fusogenic conformational changes (35) (Fig. S7) , these mAbs likely inhibit TMPRSS2 cleavage of the S2' site (via steric hindrance) and in turn activation of membrane fusion. Although residue F817 is not part of the epitope (Table S4) , the F817P substitution present in the 'HexaPro' construct likely prevents the adoption of the extended ɑ-helical conformation observed in the structures bound to VN01H1 or VP12E7 (due to restricted backbone torsion angles), resulting in dampened binding in ELISA ( Fig. 2A) .

Superimposition of the mAb-fusion peptide complexes with available prefusion S structures (PDB 6VXX) (3, 36) revealed that the targeted epitope is buried toward the core of the S trimer and is therefore inaccessible (Fig. 3, D and E and Fig. S6, B to D) , likely explaining the lack of detectable complexes of Fabs with prefusion S trimers during single particle electron microscopy analysis. Taken together, these findings suggest that the epitope recognized by fusion peptide-specific mAbs is cryptic and may become accessible only transiently (37) .

To investigate how fusion peptide-specific mAbs might bind to native S trimers and exert neutralizing activity, we transfected 293T cells to express coronavirus S proteins on the cell surface ( Fig. 4A and Fig. S8) . Interestingly, all fusion peptide-specific mAbs showed only marginal binding to SARS-CoV-2 S-expressing 293T cells, as compared to control mAbs targeting the RBD (C94) or the stem helix (C21E3). Remarkably, addition of soluble ACE2 enhanced the binding of all fusion peptide antibodies to native SARS-CoV-2 S protein to levels comparable to that of control mAbs, indicating that receptor engagement induces a conformational change that exposes the cryptic fusion peptide epitope (Fig. 4A) . Importantly, this ACE2-dependent enhancement of binding was not observed in 293T cells expressing SARS-CoV-2 S protein harboring the 2P (K986P, V987P) prefusion-stabilizing mutations (3, 36) that lay outside of the epitope ( Fig. 4A and Fig. S8) , suggesting an impediment of the receptor-induced allosteric conformational changes.

Enhanced binding of fusion peptide-specific mAbs was also observed in SARS-CoV and MERS-CoV S expressing 293T cells in the presence of the corresponding receptors ACE2 (38) and DPP4 (39) . In contrast, these mAbs bound efficiently to NL63 and 229E S 293T cells, independently of receptor engagement by ACE2 (40) or APN (41), respectively ( Fig. 4A and Fig. S8) .

It was previously shown that certain mAbs can mimic ACE2 binding and trigger fusogenic activity (42, 43) . Indeed, the addition of S2E12, an ACE2-mimicking mAb, but not S2M11, a mAb which locks SARS-CoV-2 S trimer in the closed state (42) , was also able to enhance binding of the fusion peptide antibody (Fig. 4B) . Consistent with this finding, we observed a synergy in pseudovirus neutralization when C77G12 was used in combination with S2E12, suggesting that fusion-peptide antibodies may be more effective in the context of a polyclonal response against the RBD. Collectively, these findings indicate that the fusion peptide epitope only becomes accessible upon receptor-induced S conformational changes in sarbecovirus SARS-CoV-2, SARS-CoV and merbecovirus MERS-CoV, whereas it is more readily accessible in the alphacoronavirus 229E and NL63, possibly due to molecular breathing or structural flexibility.

By screening a large number of memory B cells from immune donors using a panel of S proteins, we identified a new class of mAbs that target the fusion peptide region, some of which have unprecedented breadth being able to bind and neutralize both alpha and beta coronaviruses.

Structural analysis demonstrates that despite targeting a conserved 15 amino acid motif, these mAbs use different V genes and exhibit different binding modalities. The finding that the UCAs of these antibodies bind preferentially to common cold coronaviruses and that the antibodies acquire affinity and breadth through somatic mutations suggest that their elicitation require multiple rounds of selection, possibly by heterologous stimulation by different coronaviruses. A complex developmental pathway has been reported for HIV-1 neutralizing antibodies and may be a general requirement for antibodies that recognize highly constrained epitopes (44, 45) .

Previous studies identified serum antibodies to the fusion peptide of SARS-CoV-2 and showed, through depletion or peptide inhibition experiments, that such antibodies can contribute to the serum neutralizing activity in a polyclonal setting (29) (30) (31) (32) (33) . We show here that some fusion peptide-specific mAbs have direct neutralizing activity on pseudoviruses in vitro and reduce viral burden in vivo when administered at high doses in a hamster challenge model. Although the neutralizing activity of these mAbs is low when used alone, it is possible that in the context of a polyclonal response they may synergize with other antibodies that favor the exposure of the fusion peptide region, as shown here with the S2E12 mAb.

Although the conformational rearrangements of the fusion peptide region enabling S2' cleavage have thus far eluded structural definition, several prior studies described the biochemical events of S2' proteolytic processing for SARS-CoV-2 (46, 47) and MERS-CoV (48, 49) . The mAbs isolated in this study provided a tool for defining an allosteric conformational change in the S protein that is triggered by receptor binding or by receptor-mimicking antibodies (42, 43) . This conformational change, which is abolished by introduction of stabilizing 2P mutations at a distant site, may be required to expose the fusion peptide region to the proteolytic cleavage by TMPRSS2 or endosomal cathepsins, resulting in the activation of the S protein fusogenic activity (28, 50) .

The finding that fusion peptide epitopes are readily accessible in alphacoronaviruses even in the absence of receptor engagement is consistent with the higher neutralizing activity of fusion peptide-specific mAbs against NL63 and 229E and suggests that such conformational change might occur spontaneously as a consequence of molecular breathing. The inclusion of the prefusion stabilizing 2P mutations implies that current mRNA vaccines may disfavor the elicitation of an antibody response to the fusion peptide region.

The fusion peptide region is highly conserved across the Orthocoronavirinae subfamily and, in view of its functional relevance, may be less prone to viral escape due to the potential fitness loss of mutants (51, 52) . Broadly neutralizing mAbs to the fusion peptide region of influenza hemagglutinin and HIV-1 gp120 have guided the design of universal vaccines against these highly variable pathogens (53) (54) (55) (56) (57) (58) (59) . Similarly, the pan-coronavirus mAbs described in this study may be used as probes for the design of immunogens that can better unmask the fusion peptide region and elicit a broadly protective antibody response. Interestingly, the same region was also found to stimulate broadly reactive CD4 T cells, providing a cue for intramolecular help in the generation of such antibodies (60).

We thank all study participants who donated blood and devoted time to our research. We thank Maira Biggiogero, Alessandra Franzetti Pellanda, Elena Picciocchi, Tatiana Terrot, Sonia Tettamanti, Tiziana Urbani, Luisa Vicari and all personnel at the hospitals and nursing homes for providing blood samples, Daniela Vaqueirinho, Sandra Jovic, Isabella Giacchetto Sasselli, Rahel Schmidt and Xinlei Xi from the Sallusto laboratory for their help with blood processing, Manfred Kopf (ETH Zurich) for providing the pLVX-puro-ACE2 transfer plasmid, Hideki Tani (University of Toyama) for providing the reagents necessary for preparing VSV pseudotyped viruses, and Marc Weisshaar (ETH Zurich) for his artistic rendition of Fig. S1 .

The study was in part funded by: 

All data associated with this manuscript are available in the main text or the supplementary materials, including FACS data gating strategy. The crystal structures will be deposited to the protein data bank (PDB). All further relevant source data that support the findings of this study are available from the corresponding authors upon reasonable request. Materials are available through materials transfer agreements (MTAs). ELISA. mAbs are grouped based on the reactivity patterns. Shown is one representative experiment out of at least 2 performed. Group 2 mAbs C21G12, C21D10, and C21E3 were described in a separate study (11) (designated P34G12, P34D10, and P34E3, respectively). EC50 (ng/ml) 

Total PBMCs isolated from Ficoll density centrifugation were plated in replicate cultures at densities of 10 4 cells per well in the presence of 2.5 µg/ml of TLR agonist R848 and 1,000 U/ml IL-2. Six days later, the specificities of the secreted IgG antibodies in the culture supernatants from each culture were screened against different HCoV S antigens in parallel (primary screening).

Cultures that exhibit cross-reactive binding patterns (shown as red well) were next isolated as CD19 + IgMand IgAto enrich for IgG-secreting memory B cell blasts (62) and cloned by limiting dilution at 0.7 cell/well. Two days post cloning, the culture supernatants of the clones underwent secondary screening with the same panel of antigens to validate their binding profiles observed during primary screening. Clones which exhibit the same binding profiles as primary screening were selected for BCR retrieval by reverse transcription followed by nested PCR reactions. Paired IGH and IGK/IGL were cloned into expression vector and transfected into Expi293 cells for recombinant antibody expression. Recombinantly expressed antibodies were tested for their specificities to the S antigens as validation. Table S2 . X-ray crystallography data collection and refinement statistics -Data in parentheses are for the highest resolution shell -Rmerge = ∑(∑|Ii − 〈I〉|/∑|I|), where the first ∑ is the sum over all reflections, and the second ∑ is the sum over all measurements of a given reflection, with Ii being the ith measurement of the intensity of the reflection and 〈I〉 the average intensity of that reflection.

-Rwork/Rfree = ∑(|Fo| − 〈|Fc|〉)/∑|Fo|, where 〈|Fc|〉 is the expectation of |Fc| under the probability model used to define the likelihood function. The sum is overall reflections. Table S3 .

Convalescent donor demographics (Fig. 1, A and D Shaded box indicates contact residues. The asterisk indicates that contacts are mediated by a water molecule.

Convalescent samples were obtained from individuals with prior SARS-CoV-2 infection, validated either by positive PCR test or by anti-spike IgG serology. Samples from vaccinated individuals (naïve or pre-immune) were collected 14-29 days after the second booster shot of Pfizer/BioNTech BNT162b2 vaccine ( Table S3 ). The study protocols were approved by the Cantonal Ethics Committee of Ticino, Switzerland (CE-TI-3428, 2018-02166; CE-TI-3687, 2020-01572). All blood donors provided written informed consent for participation in the study. Human primary cell protocols were approved by the Federal Office of Public Health (no. A000197/2 to F.S.).

For monoclonal antibodies and Fab expression and purification, Expi293 (Gibco) cells were transiently transfected with heavy and light chain expression vectors, as previously described (63) .

The mAbs S309 and S2X58 were produced by Vir Switzerland using a similar protocol (5, 6) . Inference for unmutated common ancestor (UCA)

Data were processed and analyzed using the Immcantation Framework (http://immcantation.org)

with Change-O v1.0.2. First, the sequences were annotated using IgBlast version 1.16 (65) and

IMGT as reference sequences (66) . Second, clones were assigned based on IGHV genes, IGHJ gene, and junction distance with the Change-O DefineClones function. Germlines were then reconstructed using the Change-O CreateGermlines function. Finally, the phylogenic trees of each clone with their complete UCA sequences were generated with Igphyml (67).

The SARS-CoV-2 S (D614G) ectodomain trimer beginning at Q14 with a mutated S1/S2 cleavage site (SGAR), and finishing at residue K1211, followed by a TEV cleavage, fold-on trimerization motif, and an 8× His tag was expressed and purified and described previously (68) . Three micromolar S were incubated with 3 µM of the corresponding fusion peptide Fab protein for 1 h at room temperature prior the addition of 2.6 µM of S2X58 Fab. Incubation was continued at room temperature for 1 h after which samples were diluted to 0.01 mg/ml immediately before protein was adsorbed to glow-discharged manually carbon-coated copper grids for ~30 s before 2% uranyl formate staining. Micrographs were recorded using the Leginon software (69) on a 100kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k × 4k CCD camera at 67,000 nominal magnification. The defocus ranged from 2.0 to 4.0 μm and the pixel size was 1.6 Å. Data was processed using GraphPad Prism v9.0.

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How good are my data and what is the resolution?

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Cell cultures at 3 × 10 6 /ml were transiently transfected using

Purified SARS-CoV-2 S2 in postfusion conformation, was concentrated, buffer exchanged into PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) and quantified using absorption at 280 nm. The gene to express SARS-CoV-2 S2 PentaPro in prefusion conformation was synthetized by GenScript residues 686 to 1211 fused C-terminally to a foldon trimerization domain and Histagged. SARS-CoV-2 S2 PentaPro protein was expressed and purified from 200 ml of Expi293F cells

Cells were incubated at 37ºC in a humidified 5% CO2 incubator rotating at 130 rpm. Four days posttransfection, supernatants were clarified by centrifugation at 800 g for 10 min, supplemented with 350 mM NaCl and 25 mM Tris-HCl pH 8.0, further centrifuged at 14,000 g for 30 min and passed through a 1 ml His trap HP column (Cytiva) previously equilibrated with binding buffer (25 mM Tris pH 7.4 and 350 mM NaCl). SARS-CoV-2 S2 PentaPro was eluted using a linear gradient of 500 mM imidazole

Enzyme-linked immunosorbent assay (ELISA)

Costar 96-well half-area plates with high protein binding treatment (Corning, catalog no. 3690) or custom made 384-well high-binding plates

SARS-CoV (S577A, Isolate Tor2) S protein (S1+S2 ECD, catalog no. 40634-V08B)

Briefly, each amino acid in the fusion peptide sequence K811PSKRSFIEDLLFNKVTLAD830 was substituted stepwise with all 20 main amino acids. These peptide variants and wildtype peptide, as well as HA control peptides was printed on a microarray chip in triplicate

After washing, secondary antibody goat anti-human IgG (H+L) DyLight680 (0.2 µg/ml) was incubated for 45 min at room temperature before reading on Innopsys InnoScan 710-IR Microarray Scanner. Generation of recombinant ACE2-mFc Residues 18-615 of human ACE2 (UniProtKB -Q9BYF1) were synthesized by Genscript and cloned into pINFUSE-mIgG2b-Fc2 expression plasmid (InvivoGen)

ACE2 transfer plasmid was kindly provided by Manfred Kopf (ETH Zurich). pLVX-EF1a-TMPRSS2-IRES-ZsGreen1 transfer plasmid was generated from the reference pWPI-IRES

154982). pLVX-puro-spike transfer plasmid was generated from reference pHDM-SARS-CoV-2 spike (BEI resources

Stable cell lines were generated using VSV-based lentivirus transduction. Briefly, 293T cells at 70-80% confluency in T75 flask were co-transfected with transfer plasmids encoding genes of interest (ACE2, TMPRSS2 or SARS-CoV-2 S) and packaging plasmid psPax2 and envelope plasmid pMD.2G with polyethylenimine (PEI) (at a ratio of 1:2.3 DNA:PEI) (Polysciences, catalog no. 24765-2) in Optimem

Supernatants containing lentiviral particles were harvested 36 h post-transfection, filtered through 0.22 μm filter and precipitated using 40% (W/V) PEG-8000 (Promega

71380) for 4-6 h on a shaker at 4°C, and thencentrifuged for 1 h at 1,600 g at 4ºC. The lentivirus-containing pellet was resuspended in 100 μl media and was used to transduce 293T or A549 cell lines. 293T-ACE2, 293T-S, A549-ACE2 and A549-S cell lines were selected using 10 μg/ml puromycin (InvivoGen, catalog no. ant-pr-1) 4 days posttransduction. 293T-ACE2-TMPRSS2-GFP cell line was generated from 293T-ACE2 cells by subsequent transduction of pLVX-EF1a-TMPRSS2-IRES-ZsGreen1-containing lentivral prep and sorted using BD FACSAria III. A549-ACE2-TMPRSS2 and Huh7-TMPRSS2 stable cell lines were generated using commercial lentivirus (Addgene, catalog no. 154982-LV) and selected using 10 μg/ml blasticidin

Pseudotyped virus production To produce SARS-CoV-2, SARS-CoV, MERS-CoV and 229 S pseudotyped virus, full-length spike-encoding plasmids were obtained from the following manufacturers

NR-52514) from Bei Resources; SARS-CoV-2 Omicron BA.1 (VG40835-UT)

SinoBiological; NL63 (YP_003767.1) and WIV-1 (Uniprot -U5WI05) were synthesized from

HIV-based packaging plasmids (Tat, Gag-Pol and Rev) (Bei Resources, catalog no. NR-52518, NR-52517 and NR-52519) and various spike expression plasmids using PEI in Optimem. Supernatants were harvested 36 h post-transfection and pseudotyped viral particles were precipitated as described above. To produce NL63 VSV virus, HEK-293 cells were transfected with a pcDNA3.1 expression vector encoding full-length S harboring a truncation of the 20 C-terminal residues to improve membrane transport. The day after transfection, cells were For testing inhibition of spike-mediated cell-cell fusion, A549-S and A549-ACE2-TMPRSS2 cells were stained with CFSE (Thermo Fisher, catalog no. C1157) and CellTrace™ Far Red (Thermo Fisher, catalog no. C34572), respectively, according to manufacturer's instruction. Stained cells were resuspended in complete media containing Hoechst

45 Super Plan Fluor ELWD objective, FITC and Cy5 filter and images collected with a Andor Zyla sCMOS camera. Nine fields per well were imaged and were subsequently processed with Metaxpress and Powecore softwares

SARS-CoV-2 infection model in hamsters

The variant was subjected to sequencing on a MinION platform (Oxford Nanopore) directly from the nasopharyngeal swabs; passage 2 virus on Vero E6 cells was used for the study described here. The titer of the virus stock was determined by end-point dilution on Vero-E6 cells by the Reed and Muench method (76). Live virus-related work was conducted in the high-containment A3 and BSL3+ facilities of the KU Leuven Rega Institute (3CAPS)

Mesocricetus auratus) were purchased from Janvier Laboratories and were housed per two in ventilated isolator cages (IsoCage N Biocontainment System, Tecniplast) with ad libitum access to food and water and cage enrichment (wood block). The animals were acclimated for 4 days prior to study start. Housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU

Animals were prophylactically treated 24 h before infection with VN01H1 (50 mg/kg), C77G12 (25 mg/kg and 50 mg/kg) and MGH2 (50 mg/kg) via

intraperitoneal (IP) administration. Hamsters were monitored for appearance, behavior and weight

At day 4 post infection (pi), hamsters were euthanized by IP injection of 500 μl Dolethal (200 mg/ml sodium pentobarbital, Vétoquinol SA). Lungs were collected and viral RNA and infectious virus were quantified by RT-qPCR and end-point virus titration, respectively

SARS-CoV-2 RT-qPCR

rpm, 5 min) to pellet cell debris. RNA was extracted according to the manufacturer's instructions. Of 50 μl eluate

RT-qPCR was performed on a LightCycler96 platform (Roche) using the iTaq Universal Probes One-Step RT-qPCR kit (BioRad) with N2 primers and probes targeting the nucleocapsid (72). Standards of SARS-CoV-2 cDNA

000 rpm, 5min, 4ºC) to pellet the cell debris. To quantify infectious SARS-CoV-2 particles, endpoint titrations were performed on confluent Vero E6 cells in 96-well plates. Viral titers were calculated by the Reed and Muench method (77) using the Lindenbach calculator and were expressed as

Tissue sections (5 μm) were analyzed after staining with hematoxylin and eosin and scored blindly for lung damage by an expert pathologist. The scored parameters, to which a cumulative score of 1 to 3 was attributed, were the following: congestion, intra-alveolar hemorrhagic, apoptotic bodies in bronchus wall, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular inflammation

Crystallization, data collection, structure determination and analysis

20 mg/ml were mixed with the fusion peptide (KPSKRSFIEDLLFNK, GenScript) at 20 mM and incubated for 2 h at room temperature before setting up crystallization plates. Crystals of Fabs in complex with the fusion peptide were obtained at 22°C by sitting drop vapor diffusion. A total of 100 nl complex were mixed with 100 nl mother liquor solution containing 0

VN01H1 complex); 0.2 M Calcium Acetate Hydrate, 0.1 M MES NaOH

20 % (v/v) Glycerol (C13C9 complex). Drops were equilibrated against reservoir solutions for 1 week at room temperature after which crystals were flash cooled in liquid nitrogen using the mother liquor solution supplemented with 30% glycerol as a cryoprotectant. Data were remotely recorded at the Molecular Biology Consortium beamline 4.2.2 at the Advanced Light Source synchrotron facility in Berkeley, CA. Individual datasets for each complex were processed with the XDS software package (78) and Mosfilm (79) and scaled using and SCALA or aimless (80)

Transient expression and monoclonal antibody staining of HCoV S-expressing 293 cells For transient expression of HCoV spike proteins, 293T cells were co-transfected, with plasmid encoding ZsGreen (Bei Resources, catalog no. NR-52516) and corresponding HCoV spike proteins

SinoBiological that was cloned into pHDM expression plasmid with 19 amino-acid C-terminal truncation (85), using PEI in Optimem as above. For the SARS-CoV-2 S2P mutant, K986P and V987P mutations were introduced into the wildtype backbone using Q5

Transiently transfected cells were stained the following day with mAbs conjugated using DyLight® 650 Conjugation Kit

ab201803) according to manufacturer's instructions. Dylight 650-conjugated mAbs (of indicated concentrations) were incubated with 50,000 un-trypsinized 293T-S cells per well in the presence or absence of ACE2-mFc (27 μg/ml)

20 μg/ml), DPP4-hFc (27 μg/ml) and APN-hFc