# Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

Simon Blachier, Marie-Christine Vaney, Laurine Conquet, Isabelle Staropoli, Ignacio Fernández, Emilie Giraud, Atousa Arbabian, Vincent Michel, Fruzsina Szilagyi, Salomé Guez, et al.

► To cite this version:

Simon Blachier, Marie-Christine Vaney, Laurine Conquet, Isabelle Staropoli, Ignacio Fernández, et al. Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo. Nature Communications, 2025, 16 (1), pp.10268. 10.1038/s41467-025-65144-w. hal-05395436

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1 **Title**

2

3 **Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and protects**

4 **against infection in vivo**

5

6 **Authors**

7 Simon Blachier¹, Marie-Christine Vaney², Laurine Conquet³†, Isabelle Staropoli⁴†, Ignacio

8 Fernández², Emilie Giraud⁵, Atousa Arbabian², Vincent Michel⁶, Fruzsina Szilagyi¹, Salomé

9 Guez⁵, Alix Boucharlat⁵, Jeanne Chiaravalli⁵, Jaouen Tran-Rajau⁵, Evelyne Dufour⁷, Ahmed

10 Haouz⁸, Stéphane Petres⁷, Delphine Planas⁴, Xavier Montagutelli³, Fabrice Agou⁵, Pierre

11 Lafaye⁹, Gabriel Ayme⁹†, Olivier Schwartz⁴,¹⁰†, Felix A. Rey²†, Jost Enninga¹†, Anne Brelot¹*

12 **Affiliations**

13 ¹Institut Pasteur, Université Paris Cité, Dynamics of Host-Pathogen Interactions Unit, CNRS
14 UMR3691, F-75015, Paris, France

15 ²Institut Pasteur, Université Paris Cité, Structural Virology Unit, CNRS UMR3569, F-75015
16 Paris, France

17 ³Institut Pasteur, Université Paris Cité, Mouse Genetics Laboratory, F-75015 Paris, France

18 ⁴Institut Pasteur, Université Paris Cité, Virus and Immunity Unit, CNRS UMR3569, F-75015
19 Paris, France

20 ⁵Institut Pasteur, Université Paris Cité, Chemogenomic and Biological Screening Core
21 Facility, C2RT, CNRS UMR3523, F-75015 Paris, France

22 ⁶Institut Pasteur, Université Paris Cité, Pathogenesis of Vascular Infections Unit, INSERM
23 U1225, F-75015 Paris, France

24 ⁷Institut Pasteur, Université Paris Cité, Production and Purification of recombinant Proteins
25 Platform, CNRS UMR3528, F-75015 Paris, France

26 ⁸Institut Pasteur, Université Paris Cité, Crystallography Platform, CNRS UMR3528, F-75015
27 Paris, France

28 ⁹Institut Pasteur, Université Paris Cité, Antibody Engineering Platform, CNRS UMR3528, F-75015 Paris, France

30 ¹⁰Vaccine Research Institute, Créteil, France.

31 *corresponding author: anne.brelot@pasteur.fr

32 †These authors contributed equally to this work
33

**Abstract**

The continuous emergence of antibody-escape variants of SARS-CoV-2 demands the identification of alternative methods of protection against infection that do not directly target viral proteins. Here, we generated heavy-chain-only antibody (VHHs) from an alpaca immunized with the human angiotensin-converting enzyme 2 (hACE2), the major entry receptor for SARS-CoV-2. The VHHs bind hACE2 without affecting its enzymatic activity, and two of them (B07 and B09) inhibit all SARS-CoV-2 isolates tested, including the most recent circulating variants. Their X-ray structure in complex with hACE2 showed that their epitope overlaps with the footprint of the receptor binding domain (RBD) of the SARS-CoV-2 spike on hACE2. A dimeric B07-Fc fusion construct avidly bound hACE2 with an apparent dissociation constant of 0.1 nM and inhibited infection *in vitro* with an IC50 ~ 1 nM. *In vivo* experiments using K18-hACE2 mice showed that intranasal prophylactic administration of B07-Fc conferred a dose-dependent protection against SARS-CoV-2 D614G and Omicron variants. These VHHs targeting hACE2 represent potential broad-spectrum therapeutic candidates against potential new emerging coronaviruses using hACE2 as a receptor.

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Keywords: ACE2 / COVID-19 / *in vivo* protection / SARS-CoV-2 / VHH
## Introduction

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 (coronavirus disease 2019). So far, it has infected more than 770 million people with more than 7 million deaths worldwide, even though these numbers are likely underestimated (World Health Organization – WHO, 2024). The clinical use of COVID-19 vaccines and neutralizing antibodies, both targeting the viral spike protein (S), has helped control the pandemic$^{1-3}$. However, the emergence of new variants represents a continuing threat of resurgence. The latest Omicron subvariants (KP.3, XEC) contain more than 60 mutations in S compared to the Wuhan isolate$^{4}$, resulting in antigenic escape$^{5,6}$, such that previous neutralizing antibodies became poorly protective$^{7,8}$. Constantly updating vaccines and therapeutic antibodies is therefore critical$^{9,10}$. Two strategies have been proposed to combat antigenic escape: monoclonal antibody (mAbs) cocktails or bispecific antibodies$^{11}$. While both approaches can reduce viral escape, neither is currently in clinical use due to manufacturing constraints to produce these antibodies in time. Other strategies are therefore needed to complete the arsenal against COVID-19.

SARS-CoV-2 infects cells by binding its spike protein (S) to the human angiotensin-converting enzyme 2 (hACE2), an interaction that triggers fusion of viral and cell membranes$^{12}$. ACE2, a single trans-membrane glycoprotein expressed in almost all tissues$^{13}$, is responsible for the regulation of the Renin-Angiotensin-System$^{14}$. Besides its cellular form, ACE2 also exists as a soluble enzymatically active form circulating in the plasma$^{15}$. Soluble ACE2 may indirectly be involved in SARS-CoV-2 entry$^{16}$. Using soluble derivatives of ACE2 as a “decoy” to trap the S protein is an approach being studied to avoid viral escape$^{17-19}$.

Here, we developed a strategy for the direct targeting of trans-membrane ACE2 instead of S. Because of the major role of ACE2 in regulation of blood pressure and in vascular, renal, and myocardial physiology, the challenge was to develop compounds, targeting the S-binding
surface on ACE2 without affecting its catalytic activity. The enzyme's active site and its S-binding surface are located in distinct regions of hACE2, suggesting that this approach is feasible²⁰. Monoclonal antibodies targeting ACE2 proved their efficacy in inhibiting SARS-CoV-2 infection of multiple variants²¹⁻²⁵. An alternative to mAbs for clinical application is the use of camelid single chain antibodies, in particular the variable region of their heavy-chain (also called VHH or nanobodies). These VHHs, 10 times smaller than “conventional” antibodies, retain full antigen-binding capacity, have a propensity to bind small epitopes, and are easily engineered²⁶. For example, VHHs are amenable to covalent linkage with each other or with the Fc domain, thereby increasing their valency and potency, and improving their pharmacodynamic profile in vivo²⁷⁻²⁹. These features enable the development of inhalable preventive formulations, ensuring direct delivery to the lungs, thereby conferring significant advantages to the treatment of respiratory diseases. Such treatments have been used successfully for other diseases. For example, a VHH-based therapy was approved in the U.S. and Europe in 2019 for the treatment of acquired thrombotic thrombocytopenic purpura³⁰.

In this study, we produced monomeric VHHs and a dimeric VHH fused to a human IgG1 Fc-fragment that (1) specifically bind soluble hACE2, (2) recognize hACE2 at the surface of different cell types, (3) do not alter the enzymatic activity of hACE2 neither in vitro nor in cellula, and (4) efficiently inhibit multiple variants of SARS-CoV-2 as tested in vitro (Delta, BA.1, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3; BA.2.86.1, JN.1.1, KP.3.3). These VHHs engage their 3 CDRs to compete with S binding by targeting residues primarily located in helix α1 of ACE2. Moreover, prophylactic intranasal administration of the dimeric VHH-Fc protected K18-hACE2 mice and hamsters against SARS-CoV-2.

## Results

### Selection of VHHs targeting human ACE2
We immunized an alpaca with the soluble catalytic ectodomain of human ACE2 (sACE2) and we selected three specific VHHs by phage display, designated B07, B09, and B10. Sequence analysis revealed that B07 and B09 originate from the same gene (IGHV3S53) and have the same junction region (Supplementary Fig. 1), indicating they derived from the same lineage. They underwent different somatic hypermutations (15.5% and 12.9% amino acid difference with the germline for B07 and B09, respectively) but differ by only six amino acid mutations. In contrast, B10 is encoded by a different gene (IGHC3-3) and has a totally different junction region, indicating that it was derived independently from the two other VHHs (Supplementary Fig. 1).

## VHHs B07, B09, and B10 interact with soluble ACE2

The different VHHs were expressed in fusion with His and c-myc tags at the C terminal end. We characterized the interaction between VHH and soluble hACE2 by determining association ($k_{on}$) and dissociation ($k_{off}$) rate constants using biolayer interferometry (BLI) technology (Octet HTX, Sartorius)³¹ (Fig. 1a). We immobilized ACE2-Fc on AHC biosensors and incubated them with different concentrations of VHHs. From these data, we deduced a $K_D$ value of 242 nM, 214 nM, and 21 nM for B07, B09, and B10, respectively (Fig. 1a), indicating that these VHHs bind sACE2 with moderate (B07, B09) and high (B10) affinities. We confirmed these findings using VHH-His immobilized on Ni-NTA biosensors and different concentrations of sACE2 injected as analytes ($K_D$ value of 1141 nM, 1964 nM, and 22 nM for B07, B09, B10, respectively) (Supplementary Fig. 2a).

To obtain insights into the epitopes recognized by the three VHHs, we carried out a binding competition assay using the BLI system (Fig. 1b, Supplementary Fig. 2b). B07 or B09 did not bind the immobilized ACE2 pre-bound to B09 (VHH1-B09) or B07 (VHH1-B07), suggesting that these two VHHs recognize the same, or an overlapping epitope on ACE2 (Fig. 1b). On the
contrary, B10 bound VHH1-B07 or VHH1-B09, suggesting that this VHH recognizes a different epitope than B07 and B09 (Fig. 1b). In agreement with this finding, the immobilized ACE2 pre-bound to B10 (VHH1-B10) was still able to bind B07 or B09 (Fig. 1b).

## VHHs B07, B09, and B10 bind ACE2 at the surface of cells

Next, we tested the ability of the three VHHs to bind ACE2 present at the surface of different cell lines, which express exogenous (HEK293-hACE2, A549-hACE2) or endogenous human and non-human ACE2 (IGROV-1, Vero E6) (Fig. 2, Supplementary Fig. 3). We estimated ACE2 cell surface expression for each cell line by flow cytometry using a commercial ACE2 mAb (AF933) or assessing spike binding (Supplementary Fig. 3, a-c). Then, we incubated 10 µg/ml (~ 667 nM) of each VHH with the different cell lines or with parental cells, and we used an unrelated myc-tagged VHH as negative control (ctl). The myc-tagged VHHs were detected bound to the cells' surface by flow cytometry after staining with an anti-myc antibody and a fluorescent secondary antibody (Fig. 2, Supplementary Fig. 3, d-e). The three VHHs were specifically detected at the surface of all cell lines, revealing their interaction with trans-membrane ACE2. B10 showed the highest staining in accordance with its high affinity for sACE2 defined by BLI ($K_D$ 21 nM) (Fig. 1a).

## VHHs B07, B09, and B10 do not impact the enzymatic activity of hACE2

We determined whether B07, B09, and B10 VHHs affected hACE2 function (Fig. 3). ACE2 catalyzes the cleavage of small circulating peptides, thus playing a central role in the control of the renin-angiotensin system¹⁴. We assayed the impact of 10 µM of each VHH (Fig. 3a), or different doses (Fig. 3b), on the ability of sACE2 to cleave a fluorogenic peptide substrate, Mc-Ala/Dnp where Mc-Ala fluorescence is quenched by Dnp (SensoLyte assay). ACE2 enzymatic cleavage separates the two fragments, allowing recovery of Mc-Ala fluorescence, which is
monitored at excitation/emission wavelength = 330/390 nm. We found no effect of the three VHHs on peptide cleavage (Fig. 3a, b), even at 40 µM of VHHs (Fig. 3b), contrary to the specific ACE2 inhibitor DX600, used as positive control (IC50 = 0.54 µM) (Fig. 3c). We confirmed this result *in cellula* by following peptide cleavage using HEK293-hACE2 live cells instead of soluble hACE2 (Fig. 3d). This result suggested that the interaction of B07, B09, and B10 VHHs with soluble hACE2, or cell surface hACE2, did not interfere with its enzymatic activity.

## VHHs B07 and B09 inhibit infection of multiple SARS-CoV-2 variants by competing with S binding to hACE2

We examined whether the selected VHHs may inhibit the ability of SARS-CoV-2 infected cells to form syncytia using the S-Fuse assay³². We pre-incubated S-Fuse cells (U2OS-hACE2 GFP1-10 and GFP11) with serial dilutions of VHHs, exposed them to different SARS-CoV-2 variants (Delta, BA.1, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3; BA.2.86.1) and quantified syncytia formation 18 h after infection.

B07 and B09, which are closely related (Supplementary Fig. 1), inhibited syncytia formation induced with the 7 SARS-CoV-2 variants tested with similar IC50 (from 1.45 to 30 µg/ml corresponding to 95.1 to 1937 nM) (Fig. 4a, Supplementary Table 1a, b). In contrast, B10 which has the highest affinity for ACE2 (Fig. 1a), displayed no inhibition of syncytia formation induced by the different variants tested (Fig. 4a). This result is in line with B10 targeting a different epitope on hACE2 than B07 and B09, most likely not involved in S recognition.

To assess whether B07 and B09 inhibited SARS-CoV-2 infection by inhibiting binding of the S protein, we performed competition experiments in which binding of soluble S (Wuhan isolate) on HEK293-hACE2 cells was measured in the presence of increasing concentration of VHHs B07 and B09. B10 was used as control. S binding was assessed after immuno-staining with an
anti-S antibody and flow cytometry (Fig. 4b). Pre-incubation of HEK293-hACE2 with VHHs B07 and B09 prevented soluble S binding, while B10 had no effect (Fig. 4b). This suggested that B07 and B09 inhibited SARS-CoV-2 infection by inhibiting ACE2-spike interaction.

We then performed competition experiments with B07 or B09 using increasing concentrations of S from 5 to 10 µg/ml (Fig. 4c). The relationship between the observed IC50 value and tracer concentration (here S) in displacement experiments indicates whether binding inhibition is competitive (i.e. displaceable) or noncompetitive³³. The IC50 value is expected to increase linearly with S concentration for competitive inhibition³³. IC50 values indeed yielded a linear regression with increasing concentration of S (Fig. 4d), indicating that both B07 and B09 inhibited S binding through a competitive mechanism.

Structural characterization of the VHHs /sACE2 interaction

We undertook structural studies to define the epitopes of the VHHs and the inhibitory mechanism of B07. We obtained crystals of a ternary complex of the soluble, monomeric protease domain of hACE2 and VHHs B07 and B10 that diffracted at 2.7Å resolution (Fig. 5a, b, Supplementary Table 2, Supplementary Fig. 4a). The X-ray structure revealed that B10 buries, from the surrounding solvent, an area of 601 Å² of the hACE2 surface (Supplementary Fig. 4b, c), recognizing a negatively charged surface in loops β1/β2 and helices α5/6/11 (Fig. 5a, Supplementary Fig. 4d, e, Supplementary Fig. 5, Table 3), away from the RBD footprint (Supplementary Fig. 6a-c), explaining why B10 did not inhibit SARS-CoV-2 infection. We validated the epitope by introducing the E160A mutation in hACE2 helix α6, thereby breaking the negatively charged patch. As predicted, E160A mutant did not bind B10 (Supplementary Fig. 4f). B07 binds mainly to hACE2 helix α1, burying a surface area of 662.1 Å² from solvent (Fig. 5c). It forms multiple polar and van der Waals interactions via the CDR1, 2, and 3 of B07 and hACE2 helix α1 (Fig. 5c-e, Supplementary Table 4, Supplementary Fig. 5). Residues
from hACE2 helix α2 and loop β4/β5 (residue 353) also contribute to the interaction (Fig. 5c,
e; Supplementary Table 4, Supplementary Fig. 5). From the 6 amino acid differences
between B07 and B09, only two of them are in the paratope region, and one of these was a
change from Ile (germline) to Thr (B07) or Ser (B09). Except for position 60, in which a somatic
mutation from Ala to Pro took place only in B07, the remaining eight somatic mutations in the
paratope gave rise to the same substitution in both VHHs (Supplementary Fig. 1), strongly
indicating B07 and B09 recognize the same epitope. We validated the structure and B07 epitope
by substituting the critical residues H34, E35, and D38 (Fig. 5f, Supplementary Fig. 7a). The
respective mutants (H34A, E35A, D38A) abrogated B07 binding on hACE2 expressing cells
(Fig. 5f), breaking a salt bridge (E35A) or hydrogen bonds (H34A, D38A). These mutants also
inhibited the binding of B09 with cell surface hACE2 (Supplementary Fig. 7b), corroborating
that B09 shares the epitope with B07. The calculated surface electrostatic potential indicated a
strong charge complementarity (Fig. 5g). Superimposition of the hACE2/B07/B10 complex
model with the previously reported RBD/sACE2 model (PDB 6M0J) on hACE2 revealed steric
clashes between B07 (purple) and the RBD of SARS-CoV-2 (green), when bound to their
respective epitope (Fig. 5h, i, Supplementary Fig. 6a-c). This demonstrates that they cannot
bind simultaneously and explains the ability of B07 to inhibit the interaction of the spike with
hACE2. Nine residues (underlined in Supplementary Fig. 6b, in red in Supplementary Table
5) of hACE2 were involved in direct contact with both B07 and the RBD. These findings
explained the results of competition assays between VHHs and S (Fig. 4b-d). Comparison of
B07 epitope with the RBD footprints on ACE2 of various coronavirus using ACE2 as a receptor
(including, in addition to the sarbecoviruses, the merbecovirus NVHKU5R³⁴ and the
alphacoronavirus HCoV-NL63³⁵) (Supplementary Fig. 6d, e; Supplementary Table 5),
revealed a substantial overlap. These observations indicate that B07 broadly competes with the
binding of multiple coronaviruses. Finally, aligning the ACE2 amino acid sequences from
different species showed that there is a phylogenetic diversity at ACE2 positions 30, 31, 34, 35, 37 (Supplementary Fig. 5), suggesting that B07 may be impaired in its binding ability with hamster and mouse ACE2.

## VHH B07 dimerization potentiates its ability to inhibit hACE2-mediated coronavirus entry

We explored the effect of bivalent VHH variant by making a dimeric form²⁷ with VHH B07 in fusion with the human IgG1 Fc segment (i.e. hinge plus CH2 and CH3 domains)³⁶. We tested the resulting B07-Fc for its ability to (i) interact with soluble hACE2 *in vitro*, (ii) bind membrane ACE2 using different cell types, (iii) prevent the enzymatic activity of hACE2, and (iv) inhibit SARS-CoV-2 variants (Fig. 6).

B07-Fc bound soluble hACE2 with higher avidity (Fig. 6a) compared to monomeric B07 (Fig. 1), with a measured apparent $K_D$ ($K_D$ App) of 0.11 nM. B07-Fc was specifically detected at the surface of different cell lines expressing exogenous (HEK293-hACE2, A549-hACE2) or endogenous ACE2 (IGROV-1, Vero E6) by flow cytometry after staining with anti-IgG antibodies (Fig. 6b, c, Supplementary Fig. 8a, b). B07-Fc also specifically stained primary human nasal epithelial cells (hNECs), as shown by confocal immunofluorescence imaging (Fig. 6d). This air-liquid culture system is an effective tool for studying SARS-CoV-2 infection³⁷. As described³⁸,³⁹, hACE2 co-localized with α-tubulin on the surface of motile cilia (Supplementary Fig. 8c). As seen with B07, B07-Fc did not affect enzymatic peptide cleavage activity of soluble and cell surface hACE2 (Fig. 6e, f) and did not impact cell viability, even at high concentration (Supplementary Fig. 8d). Finally, B07-Fc inhibited syncytia formation induced with the 9 SARS-CoV-2 variants and sub-variants tested (Delta, BA.1, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3, BA.2.86.1, JN1.1, KP.3.3)⁵ (Fig. 6g, Supplementary Table 1c) with an IC50 at least 2 log lower than that of B07 (IC50 B07-Fc = from 0.96 to 4.15 nM versus
IC50 B07 = from 95.1 to 1937.2 nM). B07-Fc also inhibited infection of Delta and Omicron variants as measured by viral N protein detection (IC50 B07-Fc = 0.06 and 0.07 nM for JN1.1 and KP.3.3, respectively) (Fig. 6h, Supplementary Table 1c) and by RT-qPCR (IC50 B07-Fc from 7.9 to 22.4 nM for Delta, BA.1, BQ.1.1, XBB.1.5, XBB.1.16.1) (Supplementary Fig. 8e). These results suggested that the dimerization of the VHH B07 increased its inhibitory efficacy without affecting hACE2 enzymatic activity. Therefore, this dimeric VHH represented a good candidate for further *in vivo* studies.

## Genetic variation of the ACE2 N-terminal region impacts VHH B07-Fc binding efficacy

To evaluate whether human ACE2 (hACE2) polymorphisms affect B07-Fc binding efficacy, we constructed polymorphic hACE2 variants by substituting residues located within the vicinity of S and B07 binding (S19P, I21T, K26R, T27A, H34N, E35K, E37K, K68E, M82I, P84T) in a myc-hACE2 expressing plasmid. Then, we tested their cell surface expression after anti-myc staining and their reactivity with B07-Fc by flow cytometry. None of the substitution abrogated cell surface expression of the myc-hACE2 constructs (Supplementary Fig. 9a). Eight out of ten constructs were detected by B07-Fc, suggesting that hACE2 polymorphism might have little impact on B07-Fc antiviral efficacy (Fig. 6i). However, B07-Fc failed to detect hACE2-H34N and hACE2-E35K expressing cells (Fig. 6i), confirming the importance of the residues H34 and E35 for B07-Fc binding to hACE2 (Fig. 5).

Phylogenetic diversities in ACE2 might also influence B07-Fc binding (Supplementary Fig. 5). B07-Fc poorly recognized hamster and mouse ACE2 expressing cells compared to hACE2 expressing cells (Fig. 6j). BLI experiments confirmed that B07-Fc bound poorly to the soluble hamster ACE2 compared to human ACE2 ($K_{D App}$ haACE2 = 78 nM versus $K_{D App}$ hACE2 = 0.11 nM) (Fig. 6k). The presence of a glutamine at position 34 (Q34) in these different species (Supplementary Fig. 5, Supplementary Fig. 9b) likely explain the impaired binding efficacy.
Indeed, B07-Fc did not react with hACE2-H34Q (Supplementary Fig. 9c). In contrast, B07-Fc showed stronger binding for haACE2-Q34H than for haACE2-WT (Supplementary Fig. 9c). These results confirmed that B07-Fc epitope was critically dependent upon residues at position 34 of ACE2. Since residues at position 30-31 are also part of the epitope (Fig. 5c, Supplementary Table 4), it is likely that the presence of two asparagines at position 30-31 in mouse ACE2 (Supplementary Fig. 9b) explains the lower binding efficacy of B07-Fc to mouse ACE2 compared to hamster ACE2 (Fig. 6j).

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## B07-Fc protects against SARS-CoV-2 infection in vivo

We evaluated in vivo the prophylactic potential of VHH B07-Fc using the hACE2 transgenic mouse model (K18-hACE2) and XBB.1.5 Omicron variant infection. Mice treated intranasally with a single administration of VHH B07-Fc (7 mg/kg), or VHH-Fc control (7 mg/kg) were infected 24 h later intranasally with 1x10⁵ PFU of XBB.1.5 variant. Animals were euthanized at day 3 post-infection and lung viral load was measured by RT-qPCR and a conventional plaque assay (Fig. 7a-c).

In 6 of the 10 B07-Fc-treated mice, lung viral load level was strongly reduced (> 3 log) compared with the mean of the control group or below the detection limit, suggesting that the lung had been protected from viral infection (Fig. 7b, c). We assessed by immunofluorescence SARS-CoV-2 infection and the cytopathic effect induced by XBB.1.5 in the presence (B07-Fc) or absence (ctl-Fc) of B07-Fc (Fig. 7d). Nasal, respiratory, and lung epithelia were stained at day 3 p.i. with phalloidin (to visualize F-actin), anti-alpha tubulin antibodies (to visualize cilia), anti-SARS-CoV-2 N antibodies (to visualize viral replication), and DAPI (to visualize the nucleus). Treated mice showed no viral replication and no tissue damage (disappearance of the ciliated structure) compared to control mice (ctl-Fc and uninfected K18-hACE2) (Fig. 7d, Supplementary Fig. 10), corroborating the protective effect of B07-Fc. The dose of B07-Fc in

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the lung homogenate measured by ELISA was variable between treated mice, revealing probable heterogeneity in the biodistribution of VHH in the lung (Fig. 7e). Notably, all mice in which the B07-Fc dose in the lungs were above 215 ng B07-Fc/g of lung had lung viral load below the limit of detection, while the mice with lower B07-Fc dose exhibited high lung viral load (Fig. 7e). Therefore, we concluded that B07-Fc, when used prophylactically in K18-hACE2 and when efficiently delivered into the lungs, strongly protects against Omicron pulmonary infection.

To further confirm the protective effect of B07-Fc *in vivo*, we investigated the efficacy of B07-Fc in preventing lethality in K18-hACE2 mice infected with the D614G variant. Mice were treated intranasally with a single dose of B07-Fc (5 mg/kg) or VHH-ctl (5 mg/kg) and infected 24 h later with $1 \times 10^4$ PFU of the D614G variant. Body weight loss and signs of disease were measured starting from day 3 post-infection (p.i.) (Fig. 7f). All mice in thectl-Fc group reached humane endpoints (body weight loss > 20%, clinical score > 6) at day 6 p.i. and were euthanized (Fig. 7g). Mice in the B07-Fc-treated group were also sacrificed on day 6 p.i. for measuring B07-Fc concentration in the lung as in Fig. 7e (Fig. 7h). All treated mice showed delayed body weight loss and pathological scoring, although this delay was variable between treated mice (Fig. 7g). The four mice with B07-Fc concentrations in the lung homogenate reaching > 200 ng B07-Fc/g of lung (VHH_high group) showed moderate symptoms with weight loss < 10%. On the other hand, mice with a low B07-Fc concentration in the lung (VHH_low group) experienced body weight loss and symptoms similar to those of the ctl-Fc group (Fig. 7g, h). The body weight curve of the VHH_high group was significantly delayed compared with that of the ctl-Fc group (p = 0.00378 using a linear mixed model). These results suggest that B07-Fc was able to protect mice when efficiently delivered to the lung (above a threshold of around 200 ng B07-Fc/g of lung).
We also evaluated the prophylactic potential of VHH-B07-Fc using the Syrian hamster preclinical model. VHH-B07-Fc was administered intranasally at 5 mg/kg and infected 24 h later with $1 \times 10^4$ PFU of the XBB.1.16.1 variant. Lung viral load was modestly but significantly lower in hamster treated with B07-Fc compared with the control animals ($3.49 \times 10^7$ vs $5.7 \times 10^8$ copies/g lung) (Supplementary Fig. 11), indicating a lower protection potential of VHH-B07-Fc in this species compared with transgenic mice expressing human ACE2.

## Discussion

Anti-SARS-CoV-2 monoclonal antibodies (mAbs) used for prophylaxis or therapeutic treatment are losing their efficacy in the face of the continuous emergence of resistant variants⁷. Therefore, targeting ACE2 represents an alternative to the use of mAbs targeting the spike (S) protein. In line with previous studies²¹⁻²⁵, we found that anti-ACE2 conferred inhibition against a broad spectrum of variants (Fig. 4 and 6). We described here anti-hACE2 VHHs inhibiting infection by 9 variants, including the most recent Omicron subvariants (Delta, BA.1, BQ1.1, XBB.1.5, XBB.1.16, EG.5.1, BA.2.86, JN.1.1, KP.3.3). In comparison, the therapeutic anti-S mAb sotrovimab lost antiviral activity against BA.2.86.1 and JN.1$^{40}$⁵. Similarly, the mAbs pemivibart and sipavibart remained poorly active or lost efficacy against KP3.3⁷, underscoring the pertinence of our host-targeting approach.

An issue with targeting host proteins is the potential loss of physiological functions of the target. Here, the anti-ACE2 VHHs bind the peptidase domain of hACE2 (Fig. 5) without interfering with its peptidase activity, as showed both *in vitro* and *in cellula* (Fig. 3, Fig. 6e, f). They inhibit SARS-CoV-2 infection by competitively blocking the interaction with S (Fig. 4b-d), targeting an epitope comprising residues mainly located in hACE2 helix α1 (Fig. 5), which are also recognized by the viral RDB (Supplementary Fig. 6). This epitope, distal to the enzyme's active site (Supplementary Fig. 5), should not induce side effects if it is targeted, supporting
349 the translational potential of these antibodies. An additional advantage is that the emergence of
350 escape mutants after treatment with competitive anti-ACE2 VHHs is unlikely.

351 Only low-frequency polymorphisms in hACE2 (H34N and E35K, p ~ 10⁻⁵)⁴¹ abolished B07-Fc
352 binding, suggesting that variations in hACE2 should have little effect on the VHH efficacy. We
353 note that these two hACE2 variants may also block RBD binding (Supplementary Fig. 6)⁴².

354 Structural comparisons of the footprint of the RBDs of SARS-CoV and HCoV-NL63 revealed
355 that B07-Fc is also expected to block these two related viruses, thus expanding the scope of its
356 activity. We also compared the footprint of B07 on ACE2 with the one of NVHKU5R on mink
357 ACE2. Indeed, mink has been identified as a reservoir for coronaviruses⁴³. The epitope of
358 NVHKU5R on mink ACE2 overlaps with the one of B07 on hACE2 (Supplementary Fig. 6d,
359 e), suggesting that B07-Fc will also block potential emerging human-adapted coronavirus
360 derived from mink.

361 Our VHHs have the capacity to rival previously described anti-hACE2<sup>21-23</sup> B10, which is not
362 inhibitory but binds to hACE2 with high affinity (K<sub>D App</sub> = 21 nM), represents a valuable tool
363 for tracking hACE2 in SARS-CoV-2 infected cells and/or detecting hACE2 in cryptic primary
364 cell locations that are not accessible to conventional antibodies. B07-Fc exhibited high affinity
365 for hACE2 (K<sub>D App</sub> = 0.11 nM) and potent efficacy in inhibiting SARS-CoV-2 infection (IC50
366 ~ 1 nM corresponding to ~ 5 ng/mL). It is one order of magnitude more potent than the most
367 promising mAbs anti-hACE2 05B04 (IC50 ~ 50 ng/mL), when evaluated in the same infection
368 inhibition assay<sup>22</sup>. Both B07-Fc and 05B04 act by a competitive mechanism, engaging hACE2
369 residues also targeted by the RBD (residues 27-42 for B07-Fc; residues 19-31 for 05B04<sup>22</sup>)
370 (Supplementary Fig. 6). Compared to conventional mAb-based therapies<sup>21-23</sup>, VHH-based
371 therapies have several advantages. VHHs are smaller in size than mAbs, even when fused to a
372 Fc fragment (~80 kDa for VHH-Fc vs ~150 kDa for IgG), and they show improved tissue
373 penetration<sup>26,27</sup>. Their single-gene format is also simpler to engineer, reducing production cost.
VHHs could be engineered into a multi-specific format by combining two or three VHHs in a
single polypeptide to target distinct epitopes or even distinct receptors<sup>44</sup>, potentially with
synergistic effects<sup>45</sup>. We found that engrafting B07 onto the Fc domain of conventional IgG1
results per se in a dimeric VHH with highly increased inhibitory efficacy (<strong>Fig. 6g, h</strong>), while
showing no toxicity on cells (<strong>Supplementary Fig. 8d</strong>). The fusion with conventional Fc
domain allows VHHs to bind Fc receptors, thereby rescuing them from degradation *in vivo*<sup>29</sup>,
which improves their pharmacokinetic profile<sup>27,28</sup>. However, Fc fusion may activate Fc effector
function in immune cells or with complement, or cause antibody-dependence enhancement
(ADE)<sup>46</sup>. To mitigate these risks and increase the potential of therapeutic properties, an optimal
balance of Fc effector functions can be achieved by introducing point mutations in the Fc
domain<sup>47,48</sup>. It would be of interest to explore the effect of known mutations on the efficacy of
VHH B07-Fc.

Without additional development in the formulation, VHH B07-Fc is already highly stable to be
administrated by inhalation<sup>49,50</sup>. B07-Fc prevented the infection of human ACE2-expressing
mice (K18-hACE2) by D614G or Omicron XBB.1.5 following intranasal administration (<strong>Fig. 7</strong>). The observed heterogeneity in the number of fully protected mice correlated with the
effective concentration of B07-Fc in the lung, likely reflecting a sub-optimal distribution of
B07-Fc. Although the administration procedures require optimization to further boost overall
protection<sup>51</sup>, beyond a threshold dose of B07-Fc in the lung (about 200 ng of B07-Fc/g lung),
no viral load was detected in animals. We confirmed this protective effect of B07-Fc after
intranasal administration in the preclinical Syrian hamster model<sup>52-54</sup> (<strong>Supplementary Fig. 11</strong>),
although this protection was reduced. The weak binding of B07-Fc to hamster ACE2 compared
to hACE2 (<strong>Fig. 6k</strong>) likely explains the low efficacy of B07-Fc treatment in hamsters.
Intranasal administration of anti-S VHHs has already proved its efficacy to prevent SARS-
CoV-2 infection in animal models<sup>55-57</sup> and to reduce the viral load in the brain<sup>58</sup>. In the case of
anti-ACE2, this mode of administration, by promoting bio-distribution in the lung, may also
overcome a loss of anti-ACE2 activity due to the wide expression of ACE2 in different tissues
and the presence of circulating ACE2 in the blood¹⁵. This feature could result in the use of much
reduced dose to achieve antiviral activity. In addition, respiratory drug delivery presents low
logistical burden and possible self-administration, which would be particularly beneficial in the
case of SARS-CoV-2⁵⁵.

Overall, our anti-ACE2 VHHs provide a potential solution to the never-ending escape of SARS-CoV-2 from anti-S antibodies, offering novel opportunities for therapeutic treatments and prophylaxis.

408

409 Methods

410 Expression and purification of hACE2 peptidase domain

For alpaca immunization the ACE2 peptidase domain (residues 19-615, Uniprot: Q9BYF1) was
cloned in pcDNA3.1. The signal peptide from the IgK chain
(METDTLLLWVLLLLWVPGSTG) was introduced at the N-terminus to ensure the protein
secretion to the extracellular medium, and His(x8) and Strep tags were added *in-tandem* at the
C-terminus, following a Thrombin cleavage site. For structural studies, the Strep tag was
exchanged for the ybbR tag (DSLEFIASKLA).

The plasmid coding for ACE2-His-Strep was transiently transfected into Expi293F™ cells
(Thermo-Fischer) using FectoPro® DNA transfection reagent (PolyPlus) according to the
manufacturer's instructions, and 5 µM kifunensine was added to the culture medium
immediately after transfection. The cells were incubated 5 days at 37 °C and harvested by
centrifugation. The supernatant was concentrated and recombinant hACE2 was purified by
affinity chromatography on a Streptactin column (IBA) and by size-exclusion chromatography
(SEC) on a Superdex200 column (Cytiva) equilibrated 10 mM Tris, 100 mM NaCl, pH8.
hACE2 was digested over-night at room temperature with Thrombin (1 U per 100 µg of recombinant protein) to remove the His and Strep tags. The proteolysis was stopped with 1 mM PMSF (phenylmethylsulfonyl fluoride) and the untagged ACE2 was recovered in the flow-through after injection in a Streptactin column. A final SEC was performed, after which untagged ACE2 was concentrated and stored at -80 °C. For VHH epitope mapping experiments using BLI, ACE2 peptidase domain fused to a human Fc from IgG³⁶ was used (sACE2-Fc). For the measurement of the affinity between VHH B07-Fc and sACE2 by interferometry (BLI), we used a His-tagged sACE2 (sACE2-His) from human or hamster species (R&D systems).

For structural studies, the ACE2-His-ybbR plasmid was transfected into Expi293F™ GnTI-cells (Thermo-Fischer) using FectoPro® DNA transfection reagent (PolyPlus) according to the manufacturer's instructions. The cells were incubated 5 days at 37 °C and harvested by centrifugation. The supernatant was concentrated and recombinant ACE2 was purified by IMAC on a His-Excel column. The eluted protein was concentrated, the buffer was exchanged (10 mM Tris-HCl, 100 mM NaCl, pH 8.0) and ACE2 was digested over-night at room temperature with Thrombin (1 U per 100 µg of recombinant protein). The cleavage was stopped with 1 mM PMSF (phenylmethylsulfonyl fluoride) and untagged ACE2 was recovered in the flow-through fraction after injection on the His-Excel column. The protein was concentrated and incubated over-night at room temperature with 1 U of endoglycosidase H in acidic buffer (NaAc 100 mM, NaCl 100 mM, pH 5.5). A final SEC was performed using a column equilibrated with 10 mM Tris-HCl, 100 mM NaCl, pH 8.0.

### Plasmids, site-directed mutagenesis, and transfection

Mouse ACE2 plasmids was purchased from addgene (pscALPSpuro-MmACE2, #158087).

Hamster ACE2 pcDNA3.1 hygro plasmid was synthetized by GeneArt (ThermoFisher). The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-
hACE2-BSD³². The construct myc-hACE2 (myc-hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with myc-hACE2 from the pCEP4-myc-ACE2 (addgene, #141185)⁵⁹. Polymorphism substitutions and alanine substitutions were generated by site-directed mutagenesis using the Q5-site directed mutagenesis kit (New England Biolabs) according to the manufacturer's instructions. The mutants were all verified by sequencing (Eurofins). For transient expression, HEK293 (2 x 10⁵) cells were cotransfected (lipofectamine 2000) with WT or mutant ACE2 vectors and with eGFP-N1 (Clontech, Palo Alto, Calif.), in a 5:1 ratio. The eGFP positive cells were analyzed by flow cytometry after immunostaining.

## Viruses

SARS-CoV-2 Delta (B.1.617.2; GISAID ID: EPI_ISL_2029113), D614G (hCoV-19/France/GE1973/2020; GISAID ID: EPI_ISL_414631), Omicron BA.1 (GISAID ID: EPI_ISL_6794907), BQ.1.1 (GISAID ID: EPI_ISL_15731523), and JN.1.1 (hCoV-19/France/HDF-IPP21391/2023) were previously described⁵,⁴⁰,⁶⁰,⁶¹. XBB.1.5 was isolated from a nasopharyngeal swab of an individual of Hôpital Européen Georges Pompidou (HEGP; Assistance Publique, Hôpitaux de Paris) (GISAID ID: EPI_ISL_16353849)⁶². The laboratory of Virology of HEGP sequenced the swabs. The patient provided informed consent for the use of the biological materials. The XBB.1.16.1 strain (hCoV-19/France/GES-IPP07712/2023), the EG.5.1.3 strain (hCoV-19/France/BRE-IPP15906/2023), and the BA.2.86.1 strain (hCoV-19/France/IDF-IPP17625/2023) were supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and headed by Dr Etienne Simon-Lorière⁵. The human sample from which strain hCoV-19/France/GES-IPP07712/2023 was isolated has been provided by Dr Vanessa COCQUERELLE from Laboratory Deux Rives. The human sample from which strain hCoV-19/France/BRE-IPP15906/2023 was isolated has been provided by Dr F. Kerdavid from Laboratoire Alliance Anabio, Melesse. The human sample
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from which hCoV-19/France/IDF-IPP17625/2023 was isolated by Dr Aude LESENNE from
Cerballiance, Lisses (France). Titration of viral stocks was performed on Vero E6 or IGROV-
1 cells, with a limiting dilution technique enabling the calculation of the median tissue culture
infectious dose or on S-Fuse cells. KP.3.3 strains (hCoV-19/France/BFC-IPP06087/2024) were
isolated and amplified by the Reference center for Respiratory Viruses hosted by Institut
Pasteur ⁷.

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480

In replication experiments, XBB.1.16.1 (hCoV-19/France/GES-IPP07712/2023), XBB.1.5 (hCoV-19/France/PDL-IPP58867/2022), BQ.1.1 (hCoV-19/France/IDF-IPP50823/2022) and BA.1 (hCoV-19/France/PDL-IPP46934i/2021) were supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). The human samples from which strains were isolated has been provided from the Laboratoire Deux Rives, CH Laval and Laboratoire des centres de santé et d'hôpitaux d'IDF. Viral productions were performed on Vero-TMPRSS2 cells and titration of viral stocks was performed on Vero E6 cells. Viral concentrations were determined through Plaque Assay and the titers were quantified as plaque forming units (pfu) per milliliter.

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The sequence of the viral stocks was verified by RNAseq by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France). All work with infectious virus was performed in biosafety level 3 containment laboratories at Institut Pasteur.

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492

Cell lines

493

HEK293-hACE2 (human embryonic kidney cell line) were generated by transfection and cultured in Blasticidin (10 µg/mL). U2OS (human osteosarcoma cell line) and A549 (human lung epithelial cell line) cells stably expressing hACE2, and U2OS-hACE2 cells stably expressing the GFP split system (GFP1-10 and GFP11; S-Fuse cells) were previously described ³². These cells were cultured in DMEM or in F-12K Nutrient Mixture Media (A549) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptavidin. Blasticidin

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498
(10 µg/mL) and puromycin (1 µg/mL) were used to select for hACE2 and GFP split transgenes expression, respectively.

Vero E6 (Vero 76, clone E6, Vero E6, ATCC® CRL-1586TM, simian kidney cell line) was obtained from ATCC (USA) and cultured in DMEM medium (Gibco) containing 10% (v/v) FBS (Gibco) and penicillin/streptavidin (ThermoFisher Scientific). IGROV-1 cells (Human ovarian cancer cell line) were from the NCI-60 cell line panel⁴⁰ and cultured in RPMI medium (ThermoFisher) containing 10% FBS and penicillin/streptavidin. Vero E6 and IGROV-1 endogenously express ACE2.

Absence of mycoplasma contamination was confirmed in all cell lines with the Mycoalert Mycoplasma Detection Kit (Lonza). All cell lines were cultured at 37°C and 5% CO₂.

## Animal immunization and library construction

All immunization processes were executed according to the French legislation and in compliance with the European Communities Council Directives (2010/63/UE, French Law 2013-118, February 6, 2013). The Animal Experimentation Ethics Committee of Pasteur Institute (CETEA 89) approved this study (2020-27412). We subcutaneously injected an adult alpaca at days 0, 21, and 36 with approximately 150 µg of ACE2 mixed with Freund complete adjuvant for the first immunization and with Freund incomplete adjuvant for the following immunizations. A blood sample of about 150 ml of the immunized animal was collected and Peripheral Blood Mononuclear cells (PBMCs) were isolated on a ficoll discontinuous gradient.

Briefly, the vhh-encoding genes isolated from PBMCs (approximately 2 x 10⁸) were then cloned into the phagemid vector pHEN6 by using primers contained enzymatic SfiI and NotI restriction sites at the 5' and 3' ends, respectively. The size of the library was estimated at about 8x10⁷ cfu (colony-forming units). Phage display protocol was performed as described in Lafaye et al.⁶³.

About 10¹² phage-VHH diluted in PBS were used to perform three rounds of panning by using sACE2 coated on Nunc Immunotubes (Maxisorp) tubes (10 µg/ml). To increase the stringency,
524

different blocking buffers for each panning were used: 2% skimmed milk as saturating agent for the first round; 5% BSA (Bovine Serum Albumin) and Odyssey blocking buffer (LI-COR Biosciences) diluted at 1/2 for the second and third rounds.

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Following panning, individual clones were screened by standard phage-ELISA procedures using an HRP/anti-M13 monoclonal antibody conjugate. Clones were selected and their VHH-encoding DNA was sequenced by Eurofins Genomics (Ebersberg, Germany). Sequence analysis was conducted using DNA strider and multiple sequence alignment with ClustalW2 (EMBL-EBI). Out of the 192 individual clones tested in the phage ELISA, 123 were positive.

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All 123 positive clones were sequenced, revealing that 80 belonged to a dominant cluster derived from the same IGHV3S53 gene as B07 and B09. Within this cluster: B07 was found 31 times; B09 was found 23 times. Given that B07 and B09 originate from the same IGHV3S53 gene and differ by only six amino acid mutations in their sequences and 2 in their CDRs, they likely represent variants of the same lineage. B10, in contrast, was found only once and originates from a different IGHV3-3 gene, indicating a distinct evolutionary origin.

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## VHH production

539

The pHEN6 vector allows the periplasmic expression and purification of selected VHHs with a c-myc tag and His-tag at the C terminal end in frame with the VHH. Transformed *E. coli* TG1 cells expressed VHHs in the periplasm after induction with IPTG (1 mM) for 16h at 30°C. After centrifugation, cells were resuspended in PBS then lysed by polymixin B sulfate (1 mg/ml) for 1h at 4°C, the periplasmic extract being obtained after centrifugation. Purified VHHs were obtained by His pure Cobalt sepharose beads (Thermo) according to the manufacturer's instructions.

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## Dimeric VHH-Fc production

547

The coding sequences of the selected VHHs were digested with NcoI and NotI and subcloned into pFuse-huIgG-Fc2 digested with NcoI and NotI⁶⁴. *E. coli* XL1-Blue transformants were

548
obtained on 2YT agar plates containing zeocin 25 µg/ml. The plasmids coding for the recombinant proteins were purified with Nucleobond Xtra Midi Plus EF (Macherey Nagel), transiently transfected in Expi293™ cells (ThermoFisher Scientific) using Fectro PRO DNA transfection reagent (Polyplus), according to the manufacturer's instructions. Cells were incubated at 37°C for 5 days and then the cultures were centrifuged. Proteins were purified from the supernatants by affinity chromatography using a HiTrap protein A HP (Cytiva), followed by Size Exclusion Chromatography on a Superdex 75 column (Cytiva) equilibrated in PBS. Peaks corresponding to the dimeric VHH-Fc proteins were concentrated and stored at -80°C until used.

### *Biolayer interferometry (BLI)*

Equilibrium binding was performed via BLI-analysis using an Octet HTX instrument (Octet HTX, Sartorius). ACE2-Fc (or VHH-His) diluted into Sartorius's PBS working buffer containing a final concentration of 300 mM NaCl (pH 7.2) were immobilized onto Anti-hIgG Fc Capture (AHC) (or Ni-NTA) biosensors (Sartorius), which were pre-equilibrated in the same buffer. Following a baseline, ACE2-coated sensors (or VHH-coated sensors) were incubated with various concentrations of monomeric VHHs (or various concentration of soluble hACE2) for 10 min to reach equilibrium. Subsequently, biosensors were put in working buffer for 10 min to initiate dissociation. All incubations were performed at a temperature of 30°C under continuous shaking (1,000 rpm). To minimize classical surface-dependent avidity artefacts and avoid artificial multiphasic behavior, all binding experiments were performed using a range of protein loading concentrations. Optimal ACE2-Fc densities on the biosensor were determined to be 1 µg/ml for B07, B09, and B10. After minimizing avidity effects, binding data were analyzed using the Octet Software 11.0 and association/dissociation curves were globally fitted with a 1:1 binding model to determine K<sub>D</sub> apparent values<sup>65</sup>. For B07-Fc binding of human or hamster ACE2, sACE2-His (1 or 2 µg/ml, R&D Systems)-coated HIS2 sensors (Sartorius) were
incubated with various concentrations of VHH-B07-Fc (as indicated) in 50 mM Tris-HCl buffer (pH 7.5), supplemented with 0.01% P20 and 0.1% BSA. Binding curves from the association and dissociation steps were fitted using a 1:1 binding model.

To measure competitive binding to sACE2 protein between the different VHHs by BLI, we immobilized ACE2 fused to the human Fc domain (sACE2-Fc) onto Anti-hIgG Fc Capture (AHC) Biosensors (Sartorius) at 5 µg/ml for 10 minutes. A baseline step was realized by dipping sensors into the working buffer. Then, a first VHH was applied at 5 µg/ml, in order to bind the immobilized ACE2 and reach saturation. The sensor was then dipped into a well containing a mixture of the same VHH with the competitor one added at the same concentration (5 µg/ml).

Measurement of VHH binding or spike binding on ACE2 expressing cells by flow cytometry

Cells (2x10⁵) (transfected or not) were incubated for 60 min at 4°C with 10 µg/ml VHHs or 0.1 µg/ml VHH-Fc, washed twice with 2% FBS-PBS, incubated for 60 min at 4°C with anti-myc antibody (9E10, Abnova), washed twice with 2% FBS-PBS, and incubated for 60 min at 4°C with AF488 (or AF647)-conjugated anti-mouse antibody (Invitrogen) (for VHHs detection), or with the AF488 (or AF647)-conjugated anti-human IgG antibody (Invitrogen) (for VHH-Fc detection). The stained cells were washed twice, fixed in PFA 1.5% and analyzed on Attune flow cytometer NxT flow cytometer (ThermoFisher) and Kaluza software. The parental cells (HEK293, A549) or a VHH control (anti-IgE or anti-IgM or R3VQ-Fc VHHs) were used as negative controls.

To perform competition experiments of soluble spike binding at increasing concentration of VHH (between 0.01 to 100 µg/ml) were incubated with HEK293-ACE2 cells in RPMI/10% FBS. After 30 min at 37°C, 5 µg/ml, or different doses (5, 8, 10 µg/ml), of soluble trimeric spike (Wuhan)⁶⁶ was added for 30 min at 37°C. Then, immuno-staining with 5 µg/ml anti-spike monoclonal antibody mAb48 (gift of Hugo Mouquet, Institut Pasteur)⁶⁷ and Alexa-Fluor-488-
conjugated anti-human IgG antibody (Invitrogen) was performed at 4°C. Fixed cells (PFA 599
1.5%) were analyzed on Attune flow cytometer NxT flow cytometer (Thermo Fisher) and 600
analyzed as described above. 601

ACE2 enzymatic activity
---

Human ACE2 activity was evaluated using SensoLyte 390 ACE2 Activity Assay Kit (AnaSpec) 603
according to the manufacturer's protocol. To measure hACE2 activity on purified sACE2, 10 604
nM of sACE2 was incubated with B07, B09, and B10 at different concentrations before adding 605
the fluorogenic peptide substrate, MethoxyCoumarin acetic acid-Ala (Mc-Ala)/Dnp, where Mc- 606
Ala is quenched by Dnp. The protease activity of sACE2 is indicated by an increase of 607
fluorescence after peptide cleavage, and Mc-Ala recovery, monitored at Ex/Em = 330 nm/390 608
nm (Tecan SPARK) 609

To measure ACE2 activity on cells, 2.5 x 10⁴ HEK293-ACE2 cells per well (96-well black 610
plate, Greiner-BioOne) were incubated with B07, B09, B10 (>20 µM) or the control inhibitor 611
DX600 (1µM) in 40 µl complete medium without phenol red (Gibco) before adding 40 µl of 612
substrat (Mc-Ala/Dnp) diluted in PBS (SensoLyte 390 ACE2 Activity Assay kit). Peptide 613
cleavage was measure in the supernatant on Tecan SPARK plate reader. 614

SARS-CoV-2 inhibition assay: S-Fuse
---

U2OS-ACE2 GFP1-10 and GFP11 cells, also termed S-Fuse cells, become GFP+ when they 166
are productively infected by SARS-CoV-2³². Cells were mixed (ratio 1:1) (U2OS-ACE2 GFP1- 167
10 (10⁴) and GFP11 cells (10⁴)) and plated in a µClear 96-well plate (Greiner Bio-One). Serial 168
dilution of VHHs were incubated with the cells at 37°C and infected with a SARS-CoV-2 strain. 169
Eighteen hours later, cells were fixed with 4% paraformaldehyde (PFA), washed and stained 172
with Hoechst (dilution 1:10000, Invitrogen). The extent of fusion was then quantified by 175
measuring the number of objects GFP+ with an Opera Phenix high-content confocal 178
microscope (PerkinElmer) and Harmony software version 4.9 (PerkinElmer). The percentage 179
649

Individual VHHs were pre-incubated with Vero E6 cells (ten specified concentrations) 2 hours prior infection. Each plate included PBS (2 µl) and remdesivir (25 µM; SelleckChem) controls.

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After the pre-incubation time, the cells were exposed to the different variants of SARS-CoV-2 (at a pre-established multiplicity of infection: MOI of 0.07 (Delta, XBB.1.16.1), 0.27 (BQ.1.1, BA.1), and 0.33 (XBB.1.5) PFU/Vero E6 cells). After a one-hour adsorption at 37°C, the supernatant was aspirated and replaced with 2% FBS/DMEM media containing the respective compounds at the indicated concentrations. The cells were then incubated at 37°C for 48 (B.1.617.2; BQ.1.1; XBB.1.16.1; BA.1) or 66 hours (XBB.1.5). The MOI and incubation time were defined previously to obtain the same rate of viral production after infection. Supernatants were collected and heat inactivated at 80°C for 20 minutes. The Luna Universal One-Step RT-qPCR Kit (New England Biolabs) was used for the detection of genomic SARS-CoV-2 RNA through RT-qPCR using a thermocycler (QuantStudio 6 thermocycler; Applied Biosystems).

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Specific primers targeting the N-region viral gene: Forward: 5' TAATCAGACAAGGAACTGATTA Reverse: 5' CGAAGGTGTGACTTCCATG were used.

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Cycle threshold (Ct) were determined by the second derivative maximum method in the QuantStudio software. The quantity of viral genomes is expressed as Ct and was normalized against the Ct values of the negative (PBS) and positive controls (remdesivir 25 µM). Curve fits and IC50 values were obtained in Prism using the variable Hill slope model.

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### Cell viability assay

668

Cell viability assays were performed in compound-treated cells using the CellTiter-Glo assay according to the manufacturer's instructions (Promega) and a Berthold Centro XS LB960 luminometer. 3,000 cells/well of Vero E6 were seeded in white with clear bottom 384-well plates. The following day, compounds were added at concentrations indicated. PBS only and 10 µM camptotecin (Sigma-Aldrich) controls were added in each plate. After 48 h incubation, 10 µl of CellTiter Glo reagent was added in each well and the luminescence was recorded using

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673
a luminometer (Berthold Technologies) with 0.5 sec integration time. Raw data were
normalized against appropriate negative (0 %) and positive controls (100 %) and are expressed
in % of cytotoxicity.

677 *Crystallization and structure determination*

678 Purified ACE2 was incubated overnight at 4 °C with VHHs B07 and B10 at final concentrations
679 of 23 µM, 129 µM, and 129 µM, respectively. Then, a SEC step was performed to isolate the
680 complex peak from unbound fractions. The complex was purified to 7.9 mg/mL and used for
681 an initial crystallization screening trials by flowing the procedure established at the
682 Crystallography Core Facility of the Institut Pasteur⁶⁹. Briefly, crystallization drops were
683 generated by mixing equal volumes (300µL) of the protein complex and reservoir solution in
684 the format of 96 Greiner plates, using a Mosquito robot, and monitored for crystal growth by a
685 Rock-Imager. The crystallization and cryo-cooling conditions are detailed in Supplementary
686 Table 2. The crystals obtained were tested at beamlines PX1 and PX2 at SOLEIL synchrotron
687 (St Aubin, France). The best dataset collected at a resolution of 2.7 Å was indexed, integrated,
688 scaled, and merged using XDS⁷⁰ and AIMLESS⁷¹. The high-resolution limit was determined
689 using CC1/2-based cutoffs of 30%⁷². The structures of molecules were then determined by
690 molecular replacement (MR) with PHASER⁷³ using the search models of hACE2 from PDB
691 code 1R42 and alpha-fold models for VHH B07 and VHH B10⁷⁴. The asymmetric unit (au)
692 contained 3 complexes of hACE2-VHH B07-VHH B10. After reconstruction of subdomains of
693 hACE2 and of CDRs of VHHs, the models were alternatively manually corrected using
694 COOT⁷⁵ and refined using non-crystallographic symmetry and TLS parameterization
695 (Translation/Libration/Screw)⁷⁶ with BUSTER-TNT⁷⁷. The final R and free R factors are 21.9%
696 and 25.4%, respectively. The final refined models were analyzed with MolProbity⁷⁸. The data
697 collection and refinement statistics are listed in Supplementary Table 2.

698 *Structural Data analysis*
The figures of the structures were prepared using the PyMOL Molecular Graphics System, version 2.0.0 (Schrödinger LLC) (http://pymol.sourceforge.net). The polar interactions (hydrogen bonds and salt bridges), the contact residues (van der Waals) and the accessible (ASA) and buried (BSA) surface areas were assessed using ‘Protein interfaces, surfaces and assemblies’ service PISA at the European Bioinformatics Institute<sup>79</sup> (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html). The intermolecular interactions were computed using a maximal cut-off distance of 3.9Å for polar contacts (PISA distance cut-off) and 4.75 Å for van der Waals contacts (PyMol distance cut-off).

For the final analysis of contacts between molecules (containing 3 complexes), we used the chain A for hACE2, chain D for VHH B10 and chain E for VHH B07 (chains D and E have lower B-factors compared to other chains of VHHs, indicative of a more orderly disposition of atoms within the crystal). However, for all the complexes the sites of interactions between hACE2 and VHHs are ordered.

Electrostatic surfaces were computed using the PDB2PQR<sup>80</sup> and APBS<sup>81</sup> calculations software. Sequence alignments were performed using clustal W omega<sup>82</sup> and plotted using ESPript server<sup>83</sup>. The accession numbers of ACE2 sequences are: Human (UniProt ACE2_HUMAN); Hamster (UniProt A0A1U7QTA1_MESAU); Mouse (UniProt ACE2edMOUSE). The VHH sequences were analysed by Abysis (http://www.abysis.org/abysis) and IMGT<sup>84</sup> (https://www.imgt.org) websites for mapping CDR framework regions according to Kabat<sup>85</sup> and IMGT conventions, respectively. The analysis of the putative germline and somatic maturation events was done with the IMGT website.

Immunofluorescence imaging of primary human nasal epithelium cells and K18-hACE2 mouse tissues.

MucilAir™, reconstructed human nasal epithelial cells (hNECs), previously differentiated for 4 weeks, were cultured in 700 µL MucilAir™ media on the basal side of the air/liquid interface
(ALI) cultures as described⁸⁶. For imaging, cells were fixed on the apical and basal sides with 4% PFA for 15 minutes. Cells were stained O/N at 4°C with anti-ACE2 VHH-B07-Fc at 6 µg/mL revealed with a Goat anti-Human Alexa Fluor-488 secondary Ab (Jackson Immuno Research Laboratories), Phalloidin Atto 565 (Sigma-Aldrich) (F-actin staining) or α-tubulin (ThermoFisher Scientific) revealed with a Goat anti-Rabbit Alexa Fluor 555 secondary Ab (Invitrogen), and DAPI (ThermoFisher Scientific, 1mg/mL) and imaged using the LSM-700 confocal microscope (Zeiss) as described³⁷,⁸⁶.

For immunofluorescence experiments on whole-mount preparations of mouse organs, following euthanasia, samples of nasal turbinates and lungs of K18-hACE2 mice were collected and fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 24h at 4°C. Then, nasal respiratory epithelium, olfactory epithelium and lung bronchioli were finely excised and post-fixed in PBS containing 4% paraformaldehyde for 1 hour, rinsed in PBS and immersed in blocking buffer (20% normal goat serum (NGS) and 0.3% Triton X-100 in PBS) for 1 hour at room temperature, and then incubated overnight at 4 C in primary antibodies (in PBS containing 2% bovine serum albumin (BSA)) : VHH-B07-Fc (at 6 µg/mL), anti-SARS-CoV-2 nucleoprotein (N) antibody (1/200; Genetex), and rat anti-α-tubulin (1/200; ThermoFisher Scientific). After rinsing in PBS, samples were incubated for 2 hours at room temperature in PBS-2% BSA with the appropriate secondary antibodies (goat anti-human Alexa Fluor 547 (Jackson Immuno Research Laboratories); donkey anti-rat Alexa-Fluor 488 (Invitrogen)), Phalloidin Atto 647 (Sigma-Aldrich) (F-actin staining) and DAPI (ThermoFisher Scientific, 1mg/mL). Fluorescent immunolabellings were analyzed with a Zeiss LSM-700 confocal microscope.

### SARS-CoV-2 infection and treatment in K18-hACE2 mouse model

B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (stock #034860) were imported from The Jackson Laboratory and bred at the Institut Pasteur under strict specific pathogen-free conditions.
Infection studies were performed on 10 to 13 week-old male and female mice, in animal biosafety level 3 (BSL-3) facilities at the Institut Pasteur. All animals were handled in strict accordance with good animal practice. Animal work was approved by the Animal Experimentation Ethics Committee (CETEA 89) of the Institut Pasteur (project dap 210050), and authorized by the French Ministry of Research (under project 31816) before the experiments were initiated. Twenty-four hours before infection, anesthetized (ketamine/xylazine) mice were administered intra-nasally (i.n.) with either 0.9% NaCl (40 µl), control VHH (7 mg/kg or 5 mg/kg in 20-30 µl) or B07-Fc (7 mg/kg in 50 µl or 5 mg/kg in 30-50 µl). On the next day, they were again anesthetized (ketamine/xylazine) and inoculated i.n. with 1 × 10⁵ PFU of SARS-CoV-2 XBB.1.5 (hCoV-19/France/PDL-IPP58867/2022; 10 µl/nostril) or 1x10⁴ PFU of SARS-CoV-2 D614G (hCoV-19/France/GE1973/2020; GISAID ID: EPI_ISL_414631; 15 µl/nostril). Clinical signs of disease (ruffled fur, hunched posture, reduced mobility and breathing difficulties) and weight loss were monitored everyday (except weekend). Each of the four criteria received a 0-2 score and was added to a global score. Mice were euthanized when reaching 25% body weight loss or a global score of 8 or euthanized at specific time points for tissue collection. On day 3 or 6 post-infection, mice were euthanized by cervical dislocation. The trachea and upper lung lobes were fixed by submersion in 10% phosphate buffered formalin for 24-36 hours prior to removal from the BSL3 for processing and transfer in 70% ethanol. Lower lung lobes (and nasal turbinates in a subset of mice) were dissected and frozen at -80°C. Samples were homogenized in 500 µl of PBS using lysing matrix M (MP Biomedical) and a MP Biomedical FastPrep 24 Tissue Homogenizer. For lung viral load measurement, genomic viral RNA was extracted using an extraction robot IDEAL-32 (IDsolutions) and the NucleoMag Pathogen extraction kit (Macherey Nagel). Viral RNA quantification was performed by quantitative reverse transcription PCR (RT-qPCR) using the IP4 set of primers and probe (nCoV_IP4-14059 Fw GGTAACTGGTATGATTTCG and
nCoV_IP4-14146 Rv CTGGTCAAGGTTAATATAGG giving a 107bp product, and
nCoV_IP4-14084 Probe(+)TCATACAAACCACGCCAGG [5']Hex [3']BHQ-1) and the Luna
Universal Probe One-Step RT-qPCR Kit (NEB). Serial dilutions of a titrated viral stock were
analyzed simultaneously to express viral loads as PFU equivalents (eqPFU) per gram of tissue.
For plaque assay, 10-fold serial dilutions of samples in DMEM were added onto VeroE6
monolayers in 24 well plates. After one-hour incubation at 37°C, the inoculum was replaced
with equivalent volume of 5% FBS DMEM and 2% Carboxymethylcellulose. Three days later,
cells were fixed with 4% formaldehyde, followed by staining with 1% crystal violet to visualize
the plaques.

Measurement of VHH concentration in lungs homogenate by ELISA

sACE2 (2 µg/mL) were coated on polystyrene plate (ThermoScientific) over night at 4°C. After washing with PBS/Tween 0.1%, plates were incubated with B07-Fc (from 0.07 to 10 ng/mL) or homogenate of lower lung lobes in PBS 0.5% Gelatin, 0.1% Tween-20 (PGT solution) for 90 min at 37°C. A negative control (sACE2 alone) was prepared under the same conditions. Then, plates were incubated at 37°C with Peroxydase Affinipure rabbit anti Human IgG (Jackson ImmunoResearch, 1/1000) in PTG for 45 min and treated with ortho-phenylenediamine solution containing 0.1% hydrogen peroxide (H2O2) for 5 min. The reaction was stopped by adding chlorohydric acid 3M (HCl) and the absorbance of the wells was measured at 492λ in a spectrophotometer-microplate reader (Sunrise, Tecan).

ELISA plates coated with sACE2 and incubated with homogenate of lower lung lobes of B07-Fc treated animals or a range of purified B07-Fc as a control were stained with peroxidase anti-human IgG.

SARS-CoV-2 infection and treatment in Syrian golden hamster

The animals used were male Syrian golden hamsters (Mesocricetus auratus, strain
RjHan:AURA), 6 week of age (weight between 70-90 grams), obtained from Janvier
Laboratories. Golden hamsters were housed and manipulated in class III safety cabinets in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture. Animal work was approved by the Animal Experimentation Ethics Committee (CETEA) of the Institut Pasteur (project dap 210011) and authorized by the French legislation (project #21045) in compliance with the European Communities Council Directives (2010/63/UE, French Law 2013-118, February 6, 2013) and according to the regulations of Institut Pasteur Animal Care Committees before the experiments were initiated. Anesthetized animals received intranasal inoculation (5 mg/kg) of VHH-B07-Fc or VHH-ctl-Fc. Twenty-four hours after inoculation, hamsters were infected intranasally (i.n.) with 10⁴ PFU of SARS-CoV-2 XBB.1.16.1 as previously described⁶⁷.

All hamsters were followed up daily. At day 3 post-infection, animals were euthanized with an excess of anesthetics (ketamine, xylazine and laocaine) (AVMA Guidelines 2020), and samples collected. 100 mg lung fragments were ground in Lysing Matrix X tubes (MP Biomedicals) using a grinder at a speed of 6.5 m/s for 30 seconds and centrifuged at 2,000 g for 10 minutes (4°C) to clarify the supernatant.

For the evaluation of the number of SARS-CoV-2 copies in tissues by RT-qPCR, 100 µL of each grinding solution was mixed with 500 µL of Trizol to deactivate virulence. The total RNA was then extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research) and quantified. The presence of genomic SARS-CoV-2 RNA in these samples was evaluated by one-step RT-qPCR in a final volume of 5 µL per reaction in 384-well PCR plates using a thermocycler (QuantStudio 6 thermocycler; Applied Biosystems). The primers used targeted N-region viral gene: Forward: 5' TAATCAGACAAGGAACTGATTA Reverse: 5' CGAAGGTGTGACTTCCATG. Viral load quantification (expressed as RNA copy number/g lung) was assessed by linear regression using a standard curve of seven known quantities of RNA transcripts containing the N-region viral gene. All collected data were plotted and
824 analyzed using GraphPad Prism software. Mann-Whitney tests were performed for statistical
825 analysis.

826 Statistical analysis

827 Figures were generated using GraphPad Prism 10 (GraphPad software). Statistical analysis
828 was conducted using GraphPad Prism 10. Statistical significance between different groups
829 was calculated using the tests indicated in each figure legend.

830

831 Acknowledgments

832 We are grateful to Julian Buchrieser (Virus and Immunity unit, Institut Pasteur) and Erika
833 Cecon (Institut Cochin, Paris, France) for the gifts of vectors expressing ACE2 and Hugo
834 Mouquet (Laboratory of Humoral Immunology, Institut Pasteur, Paris, France) for anti-spike
835 mAb antibody. We acknowledge the Molecular Biophysics Platform (Institut Pasteur, Paris) for
836 soluble proteins quality control, in particular Bertrand Raynal, Sandrine Rosario, and Sébastien
837 Brule. We thank Gaelle Chauveau and Catherine Vivier from the Antibody Engineering
838 platform for their help with the production of VHHs. We acknowledge the National Reference
839 Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) for virus collections.
840 We thank the staff of the Animal Facility (C2RA) for the breeding and care of the K18-hACE2
841 mice. We acknowledge SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation
842 facilities, and we thank the staff of beamlines PROXIMA-1 and PROXIMA-2A for
843 assistance. The authors are grateful to the staff of the crystallography platform at the Institut
844 Pasteur for robot-driven crystallization screenings. We acknowledge the members of the
845 Direction des Applications de la Recherche et des Relations Industrielles (DARRI, Institut
846 Pasteur, Paris) involved in the project, particularly Corinne Sarrazin, Serge Pauillac and Michel
847 Perez from the patent office. We thank all the members of the Dynamics of Host-Pathogen
848 Interactions Unit (Institut Pasteur) for help with experiments and discussions, particularly
849 Geneviève Janvier. We thank Françoise Porrot and Florence Guivel-Benhassine (Virus and Immunity unit, Institut Pasteur, Paris) for virus production.

851

852 **Funding**

853 Institut Pasteur "Programme Federateur de Recherche" (PFR-6- SI-COV2 project), Urgence COVID-19 Fundraising Campaign of Institut Pasteur (FAR, JE, OS, AB).

854 Institut Pasteur DARRI/CARNOT « Maturation Program » (PL, XM, FAR, AB).

855 The French Government's Investissement d'Avenir program, Laboratoire d'excellence "Integrative Biology of Emerging Infectious Diseases" (grant ANR-10-LABX-62-IBEID) (XM, FAR, JE, OS, AB).

856 Programme Fédérateur de Recherche SARS-CoV-2 & COVID-19, PFR-4 Long Covid (XM).

857 Laboratoire d'excellence "Milieu Interieur" (JE).

858 Fondation pour la Recherche Médicale (FRM) (OS).

859 ANRS (OS).

860 The Vaccine Research Institute (ANR-10-LABX-77) (OS).

861 ANR / FRM Flash Covid PROTEO-SARS-CoV-2 (OS).

862 ANR Coronamito (OS).

863 HERA european funding (OS).

864 Sanofi (OS).

865 IDISCOVR (OS).

866 ANRS-MIE (BIOVAR and PRI projects of the EMERGEN research program) (XM, FA).

867 SB is the recipient of an MESR/Ecole Doctorale BioSPC, Université Paris Cité, fellowship; FS is the recipient of an Erasmus program. DP is supported by the Vaccine Research Institute.

870
The Opera system was co-funded by Institut Pasteur and the Région île de France (DIM1Health). Funding sources are not involved in the study design, data acquisition, data analysis, data interpretation or manuscript writing.

## Author contributions

Conceptualization: AB

Proteins productions: IF, AA, ED, PL, GA

Alpaca immunization: PL, GA

In vitro assays: SB, IS, FS, AB, JC, JT-R, PL, GA

X-ray crystallography: M-CV, IF, AA, AH, FAR

In vivo assays: LC, EG, SG

Viral strains collection: XM, FA, OS

Imaging: VM, DP

Supervision: AH, SP, XM, FA, PL, OS, FR, JE, AB

Writing—original draft: AB wrote the manuscript with contributions from all authors.

**Competing interest statement:** SB, IF, IS, FAR, PL, GA, OS, AB are designated as inventors in patent application No. WO 2025/056771 A1 filed by Institut Pasteur. The patent application covers the aspects of anti-ACE2 VHH B07, B09, B10 described in the manuscript. The other authors declare no competing interests.

**Data availability:** The datasets generated during the current study are available from the corresponding author on reasonable request. The raw data generated in this study are provided
896 in the Source Data file. Coordinates and structure factors have been deposited in the Protein
897 **Data Bank** under the accession code 9R19 (hACE2 + VHH B07 + VHH B10).

898

899 **Biological materials availability:** The unique materials used in this study are readily
900 available from the authors upon request (MTA) for academic research purposes.

901

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1083 69 Weber, P. et al. High-Throughput Crystallization Pipeline at the Crystallography Core Facility of the Institut Pasteur. *Molecules* **24** (2019). https://doi.org/10.3390/molecules24244451
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1085 70 Kabsch, W. Integration, scaling, space-group assignment and post-refinement. *Acta Crystallogr D Biol Crystallogr* **66**, 133-144 (2010).
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1087 https://doi.org/10.1107/S0907444909047374
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      <td>1100</td>
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      <td>1130</td>
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      <td>1131</td>
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      <td>Figures Legends</td>
    </tr>
    <tr>
      <td>1132</td>
      <td></td>
      <td>Fig. 1: VHHs binding on soluble human ACE2 and epitope mapping. a Kinetic analysis of B07, B09, and B10 VHHs on ACE2-Fc by BioLayer Interferometry (BLI) using various concentrations of VHHs (as indicated on the figure). AHC biosensors were used to immobilize ACE2-Fc. KD of each VHHs is indicated. One representative experiment out of at least two. b</td>
    </tr>
    <tr>
      <td>1133</td>
      <td></td>
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      <td>1134</td>
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      <td>1135</td>
      <td></td>
      <td></td>
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  </tbody>
</table>
Epitope mapping. sACE2-Fc (5 µg/mL) was immobilized onto the AHC biosensors. After a baseline step, a first VHH was applied (VHH1; 5 µg/mL). The sensor was dipped in a mixture of VHH1 and the competitor VHH2 at the same concentration (5 µg/mL). An anti-IgM specific VHH was used as control (ctl). (Top) Immobilized sACE2 pre-bound to B07 (VHH1) and VHH2 being B10, B09 or IgM (ctl). (Middle) Immobilized sACE2 pre-bound to B09 (VHH1) and VHH2 being B07, B10 or IgM (ctl). (Bottom) Immobilized sACE2 pre-bound to B10 (VHH1) and VHH2 being B07, B09 or IgM (ctl).

1143

**Fig. 2: VHHs binding on cells expressing hACE2.** Cells were incubated with the different VHHs (10 µg/ml), stained with an anti-myc antibody and a AF488-conjugated anti-mouse antibody, before being analyzed by flow cytometry. **a** B07, B09, B10 binding efficacy on HEK293-hACE2-expressing cells. Data are means ± SD of three independent experiments. Parental HEK293 cells were used as control. **b** B07, B09, B10 binding efficacy on cells expressing endogenous hACE2 (IGROV-1). Anti-IgE VHH was used as a control (ctl). Data are means ± SD of at least three independent experiments. **c** Fluorescence diagram overlays: B07, B09, B10 efficacy on cells expressing exogenous (HEK293-hACE2) or endogenous hACE2 (IGROV-1). Background (blue) corresponds to the fluorescence intensity obtained on parental cells (HEK293) or using a VHH control (anti-IgE VHH) (IGROV-1).

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**Fig. 3: Enzymatic activity of soluble and membrane hACE2.** ACE2 activity in the presence of VHHs B07, B09, B10 or absence (-) was assayed using the SensoLyte 390 ACE2 Activity Assay Kit, which measures fluorogenic peptide cleavage. **a** Soluble hACE2 (sACE2) activity in the presence of 10 µM VHHs. An ACE2 inhibitor, provided with the kit, was used as control (DX600, 1µM). Data are means ± SD of three independent experiments. Paired T test: ns, nonsignificant; ** p≤0.01.** **b** VHHs B07, B09, B10 were used at different doses (one
representative experiment out of three performed in duplicates). The mock control (buffer) was arbitrarily positioned at 10⁻¹⁴. c The ACE2 inhibitor DX600 was used at different doses. Data are means ± SD of seven independent experiments. The mock control (buffer) was arbitrarily positioned at 10⁻¹⁴. d Cell surface hACE2 activity in the presence of saturating concentration of VHHs (> 20 µM) or 1 µM DX600. Data are means ± SD of four independent experiments. Paired T test: ns, nonsignificant; ** p≤0.01.

1167

**Fig. 4: Inhibition of SARS-CoV-2 induced fusion and of spike binding on hACE2 expressing cells by anti-ACE2 VHHs.**
a S-fuse assay. Inhibition by B07, B09, B10 VHHs after infection of S-Fuse cells (U2OS-hACE2 GFP1-10 and GFP11) with different SARS-CoV-2 variants (Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5, XBB1.16.1, EG.5.1.3, BA.2.86.1). Data are mean ± SD of two independent experiments. The dashed line indicates the limit of detection.
An anti-IgM (or IgE) specific VHH was used as a control (ctl). IC50 values are indicated in the table. b Inhibition of 5 µg/mL spike binding to hACE2-expressing HEK293 cells by increasing concentrations of VHHs B07, B09 or B10. HEK293-hACE2 cells pretreated or not with the VHHs and incubated with soluble spike (S) protein (Wuhan) were stained with an anti-S antibody. Results were normalized for nonspecific (0%) and specific binding in the absence of inhibitor (100%). Experiments were fitted to a one-site competitive binding model. Data are mean ± SD of at least two independent experiments. c Displacement of S (Wuhan) binding by B07 (left) or B09 (right) for various concentrations of S (5 µg/mL, 8 µg/mL, 10 µg/mL).
Experiments were carried out as in b (one representative experiment out of at least two). d Relationship between the observed IC50 values for B07 or B09 displacement of S binding and initial concentration of S (Wuhan). Results represent means ± SD of at least two independent experiments. Linear regression analysis: R² B07=0.9005; R² B09=0.7896.

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**Fig. 5: X-ray structure of hACE2-B07-B10 complex explains the competitive binding of B07 and RBDs on hACE2.**
**a** Crystal structure of hACE2 (light blue) in complex with B07 (purple) and B10 (yellow) (PDB: 9R19). **b** Surface representation of the complex between hACE2 (light blue) and B07 (purple) rotated to 90°C from **a**. For clarity B10 was not displayed. **c** Open-book representation and footprints (in white) of B07 on hACE2 (left) and hACE2 on B07 (right). The buried surfaces (BSA) are indicated under each surface. Residues of hACE2 in red correspond to those for which alanine substitutions compromise B07 VHH binding to hACE2 mutants (**f**). **d** B07 complementarity determining regions (CDRs) colored in green (CDR1), blue (CDR2), and red (CDR3). hACE2 footprint on B07 is represented. **e** Hydrogen bonds and salt bridges (yellow dashed lines) between hACE2 and B07. **f** Impact of hACE2 alanine substitution on B07 binding. Cells were transfected with myc tagged or untagged hACE2 alanine mutants, incubated with an anti-myc antibody or B07 (10 µg/mL), respectively and stained with a mouse anti-myc antibody and a AF647-conjugated anti-mouse antibody before being analyzed by flow cytometry. Data are mean ± SD of at least three independent experiments. **g** Electrostatic potential mapped on the surface of the structure of hACE2 and B07. **h** Superimposition of the binding area of B07 (purple) and SARS-CoV-2 RBD (PDB: 6M0J, green) on hACE2 (light blue). **i** Footprints of the site of interaction of B07 and RBD on hACE2. The contours are colored in purple (B07) or in green (RBD). The common area is striped.

**Fig. 6: Fusion of VHH B07 to the Fc fragment enhances its avidity and potency.**
**a** Kinetic analysis of VHH B07-Fc on hACE2-His by BioLayer Interferometry (BLI) using various concentrations of VHH B07-Fc (indicated on the figure). HIS2 biosensors were used to immobilize 1 µg/mL of hACE2-His. **b** VHH B07-Fc binding on cells expressing exogenous (HEK293-hACE2) or endogenous hACE2 (IGROV-1). Experiments were performed as in Fig. 2 but using 0.1 µg/mL of VHH B07-Fc. Data are mean ± SD of at least four independent
experiments. **c** Fluorescence diagram overlays as performed in Fig. 2c. **d** hACE2, phalloidin (F-actin) and DAPI staining on primary human nasal epithelial cells (hNECs). Representative immunofluorescence staining of hACE2 (Red: B07-Fc staining) in combination with phalloidin (Cyan) and DAPI (Blue). Scale bars:ctl-Fc, 10 µm; B07-Fc, 5 µm. **e** Enzymatic activity of soluble hACE2 (sACE2) in the presence of VHH B07-Fc or absence using the SensoLyte 390 ACE2 Activity Assay Kit, as performed in Fig. 3. DX600 was an inhibitor control of the kit (one representative experiment out of two). **f** Cell surface hACE2 activity in the presence of saturating concentration of VHHs (10 µM) or 1 µM DX600. Data are means ± SD of three independent experiments. Paired T test: ns, nonsignificant; **p**≤0.01. **g** Inhibition of fusion (S-Fuse assay) by B07-Fc after infection of S-Fuse cells with different SARS-CoV-2 variants: Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3, BA.2.86.1, JN.1.1, KP.3.3. The dashed line indicates the limit of detection. Data are mean ± SD of at least two independent experiments. **h** Inhibition of infection quantified by counting SARS-CoV-2 nucleoprotein (N) positive cells. IGROV-1 cells were pre-incubated 1 h with serial dilutions of B07-Fc and infected with the indicated variants at 3 x 10⁻² infectious units per cell. Cells were stained with a pan-coronavirus anti-N antibody at day 1 pi. The percentage of inhibition is represented. One representative experiment performed in duplicates (**i-k**) Impact of hACE2 genetic variation on VHH B07-Fc binding. (**i**, **j**) Cells were transfected with different ACE2 constructs (**i** hACE2 polymorphism; **j** human, hamster or mouse ACE2), incubated with B07-Fc (0.1 µg/mL or different doses) and stained with an AF647-conjugated anti-human antibody before being analyzed by flow cytometry. Data are mean ± SD of at least three independent experiments. **k** Kinetic analysis of B07-Fc on hamster ACE2-His by BioLayer Interferometry (BLI) using various concentrations of VHH B07-Fc (indicated on the figure). HIS2 biosensors were used to immobilize 2 µg/mL of His-ACE2. K<sub>D App</sub> are indicated.
<figure><img src="image_2.png" /></figure>
1261 **Supplementary Figure Legends**

1262 **Supplementary Fig. 1: Sequence alignment of B07, B09, and B10 VHHs.** In dark blue 1263 background are the residues strictly conserved; in light blue background are the residues 1264 conserved for 2 sequences; in white background, residues which are not conserved. Lines above 1265 the sequences mark the CDRs in the IMGT definition (in bleu) and in the KABAT definition 1266 (in black). The sequence numbering, above and under the sequences, is according to 1267 IMGT definition and follow the numbering of the structures, respectively for B10 and B07. The 1268 contacts of B10 and B07 with hACE2 are reported under each residue as defined by the 1269 symbols in the inset. The somatic mutations are in red with germline residues in smaller font 1270 underneath. The secondary structure elements of B10 and B07 structures are indicated above 1271 each sequence. The green ovals indicate the amino acid difference between B07 and B09. J is 1272 the junction segment.

1273 **Supplementary Fig. 2: VHH binding on sACE2 and schematic of the binding competition** 1274 **assay.** **a** Kinetic analysis of sACE2 on B07-His, B09-His, and B10-His VHHs by BioLayer 1275 Interferometry (BLI) using various concentrations of sACE2 (as indicated on the figure). Ni- 1276 NTA biosensors were used to immobilize His-VHH. K<sub>D</sub> of each VHHs is indicated. One 1277 representative experiment out of three. **b** Schematic representation of the binding competition 1278 assay. Following a baseline step, the sACE2-Fc (5 µg/mL) was immobilized onto the AHC 1279 biosensors. After a second baseline step, a first VHH was applied (VHH1; 5 µg/mL). The sensor 1280 was dipped in a mixture of VHH1 + the competitor VHH2 at the same concentration (5 µg/mL).

1281 **Supplementary Fig. 3: VHHs binding on cells expressing ACE2.** **a** Example of FACS 1282 sequential gating strategy for detection of hACE2 on cell surface of HEK293-hACE2 cells by 1283 VHH or mAbs. **b** ACE2 cell surface detection by the commercial anti-ACE2 antibody AF933 1284 (R&D system, 1 µg/mL). **c** Spike binding to hACE2-expressing cells. Cells were incubated 1285 with soluble spike (S) protein (Wuhan) (10 µg/mL) and stained with an anti-S antibody. **d** B07,
1286 B09, and B10 binding on A549-hACE2 (top) and Vero E6 (bottom) cells. Cells were incubated
1287 with the VHHs (10 µg/mL), stained with an anti-myc antibody and a AF488-conjugated anti-
1288 mouse antibody, before being analyzed by flow cytometry. Parental A549 cells or myc-tag
1289 VHH IgE were used as control. Data are mean ± SD of at least 3 independent experiments. e
1290 Fluorescence diagram overlays: B07, B09, B10 efficacy on different cell lines expressing
1291 exogenous (A549-hACE2) or endogenous non-human ACE2 (Vero E6). Background (blue)
1292 corresponds to the fluorescence intensity obtained on parental cells (A549) or using a VHH
1293 control (anti-IgE VHH) (Vero E6).

1294 **Supplementary Fig. 4: Interactions between hACE2 and VHH B10.** **a** Structure of
1295 hACE2/B07/ B10 complex. **b** Rotated view from (a) as indicated and open-book representation
1296 of complex hACE2 and VHH B10 (for clarity the VHH B07 was not displayed). The surface
1297 of contacts between hAEC2 and VHH B10 are displayed in white and contoured in green. The
1298 buried surfaces (BSA) are indicated under each surface. **c** Open-book interactions as in (b) with
1299 B10 colored complementarity determining regions (CDRs) in green (CDR1), blue (CDR2), and
1300 red (CDR3). **d** Detailed interaction between hACE2 and B10 (hydrogen bonds in black dashed
1301 lines). **e** Electrostatic potential mapped on the surface of the structure of hACE2 and B10. **f**
1302 Impact of hACE2 E160A substitution on B10 binding. Cells were transfected with myc tagged
1303 or untagged hACE2 E160A (and eGFP), incubated with an anti-myc antibody or B10 (10
1304 µg/ml) and stained with a mouse anti-myc antibody and a AF647-conjugated anti-mouse
1305 antibody before being analyzed by flow cytometry. Data are mean ± SD of two independent
1306 experiments.

1307 **Supplementary Fig. 5: Sequence alignment of human, hamster and mouse ACE2.**

1308 **Supplementary Fig. 6: Interactions of hACE2 with SARS-CoV-2 RBD (PDB: 6M0J) and**
1309 **footprints of interactions in different complexes.** **a** Structure of hACE2 (pink) / RBD SARS-
1310 CoV-2 (green) (PDB 6M0J) displayed as surfaces. **b** Open-book representation and footprints
(in white) of RBD on hACE2 (left) and hACE2 on RBD (right). The buried surface (BSA) are indicated under each surface. The residues in hACE2 also involved in the interactions with VHH B07 are underlined. **c** Detailed interactions between hACE2 and RBD (hydrogen bonds in yellow dashed lines). **d** Footprints of VHH B07 and RBDs on ACE2 for a selection of viruses. Top, structures of complexes as indicated. Bottom, 90° views from top, in which the footprints (in white) of each molecule (VHH B07 and RBDs) on the top views are displayed on ACE2. **e** Each footprint is represented as an outline colored according to (d) and superposed on the hACE2/VHH B07/VHH B10 structure showing their overlap.

**Supplementary Fig. 7: Impact of hACE2 substitution on VHH binding.** **a** Example of FACS sequential gating strategy for B07 detection of hACE2 at the cell surface of HEK293 cells co-transfected with plasmids coding hACE2 and eGFP (ratio 5/1, respectively). **b** Impact of alanine substitution on B09 binding. Cells were transfected with untagged or myc tag hACE2 alanine mutants (and eGFP), incubated with an anti-myc antibody or B09 (10 µg/mL) and stained with a mouse anti-myc antibody and a AF647-conjugated anti-mouse antibody before being analyzed by flow cytometry. Data are mean ± SD of at least three independent experiments.

**Supplementary Fig. 8: B07-Fc activities.** **a** B07-Fc binding on A549-hACE2 and Vero E6 cells. Cells were incubated with 0.1 µg/mL B07-Fc and a AF488-conjugated secondary antibody, and analyzed as in Fig 6b. Data are mean ± SD of at least 3 independent experiments.

**b** Fluorescence diagram overlays performed as in Fig. 6c on A549-hACE2 and Vero E6 cells.

**c** B07-Fc detection of hACE2 on ciliated cells of human nasal epithelial cells (hNEC).

Representative immunofluorescence staining of hACE2 (Red: B07-Fc staining) in combination with a-tubulin (Green). Scale bars: left, 10 µm; right, 5 µm. **d** Vero E6 cells viability in the presence of B07-Fc measured by quantitating ATP using the CellTiter-Glo assay (triplicates).

**e** Inhibition of SARS-CoV-2 replication by B07-Fc measured by RT-qPCR. Vero E6 cells pre-incubated with B07-Fc were infected with Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5 and
1336 XBB.1.16.1 (MOI and incubation time adjusted depending on virus replication, see Methods)
1337 and viral replication was measured by quantitative RT-qPCR (one experiment in triplicates).

1338 **Supplementary Fig. 9: Relative cell surface expression of ACE2 mutants and impact of
substitutions on B07-Fc binding.** HEK293 cells transfected with plasmids encoding myc-
hACE2-WT or the indicated mutant were stained with an antibody against the myc epitope or
1340 with B07-Fc and analyzed by flow cytometry. **a** Bars represent staining efficiency for cells
1341 expressing myc-hACE2 mutants relative to cells expressing myc-hACE2-WT after anti-myc
1342 staining. Data are means ± SD of at least three experiments. **b** Alignment of the amino acid
1343 sequences of human, hamster, and mouse ACE2 from position 27 to 42. Residues involved in
1344 B07 epitope are in bold. Purple stars indicate polar interactions between hACE2 and B07. **c**
1345 Bars represent HEK293 cells transfected with plasmids encoding myc-hACE2 (top) or myc-
1346 hamster ACE2 (bottom) and stained with an antibody against the myc epitope (left) or with
1347 B07-Fc (right) and analyzed by flow cytometry.
1348

1349 **Supplementary Fig. 10: Separate color (top) or black and white (bottom) images of Fig. 7d.** Phalloidin (F-actin), SARS-CoV-2 nucleocapsid (N) (viral replication), and tubulin staining on nasal respiratory epithelium, olfactory epithelium and lung bronchioli extracted from K18-hACE2 uninfected mice, and animals that received VHH-Fc (Ctrl-Fc or B07-Fc). Representative immunofluorescence staining of phalloidin (Cyan), SARS-CoV-2 N protein (Red), and tubulin (Green). Scale bars: 10 µm.
1350
1351
1352
1353
1354

1355 **Supplementary Fig. 11: Effect of VHH B07-Fc on hamster.** **a** Schematic diagram showing
1356 the experimental design of B07-Fc prophylaxis in XBB.1.16.1 infected hamsters. Animals
1357 received intranasally (i.n.) 5 mg/kg VHH-B07-Fc (B07-Fc) or 5 mg/kg VHH-Fcctl (ctl-Fc).
1358 Twenty four hours later, they were infected with 10⁴PFU XBB.1.16.1 intranasally (i.n.). Three
1359 days post-infection, lungs were collected for analysis. Animal behavior and weight were
followed each day. **b** RNA load measured by RT-qPCR of SARS-CoV-2 in lung. Data are mean ± SD of 4 animals. Mann-Witney test: P value 0.0286*.

**Supplementary Table 1: Statistical analyses and IC50 related to Fig. 4a and 6g (S-Fuse assay).**

**a** P-values 95% confidence intervals for VHH IC50 values and R² values relative to B07 and B09 IC50s (Fig. 4a). **b** B07 IC50 values in nM (Fig. 4a). **c** P-values 95% confidence intervals and R² values relative to B07-Fc IC50 obtained in S-Fuse (Fig. 6g) or inhibition infection (anti-N) assays (Fig. 6h).

**Supplementary Table 2: Crystallization conditions, data collection and refinement statistics.**

**Supplementary Table 3: Van der Waals and polar interactions between hACE2 and VHH B10.**

CDR: complementary-determining region (Kabat definition); FR: Framework region; ss: secondary structure in hACE2; H: hydrogen bonds; S: salt-bridges; ASA: accessible surface area; BSA: buried surface area; ΔGi: solvation energy effect; Vertical bars in BSA columns indicate the buried area percentage (with one bar per 10% of ASA).

**Supplementary Table 4: Van der Waals and polar interactions between hACE2 and VHH B07.**

CDR: complementary-determining region (Kabat definition); FR: Framework region; ss: secondary structure in hACE2; H: hydrogen bonds; S: salt-bridges; ASA: accessible surface area; BSA: buried surface area; ΔGi: solvation energy effect; Vertical bars in BSA columns indicate the buried area percentage (with one bar per 10% of ASA).

**Supplementary Table 5: Van der Waals contacts between hACE2 and VHH B07 and between hACE2 and RBDs for a selection of viruses using hACE2 as a receptor.**

ss: secondary structure of hACE2; H: hydrogen bonds; S: salt-bridges; BSA: buried surface area; Vertical bars in BSA columns indicate the buried area percentage (with one bar per 10% of ASA of each indicated residue). In bold red are indicated the common interactions between B07
1384 and SARS-CoV-2 RBD structures. In bold red italic are indicated the common interactions

1385 between the four structures.
<caption>Fig. 1</caption>

a

b

B07: K<sub>D</sub>= 242 ± 8 nM

$$
K_{\text{on}} = 4.15 \times 10^{-4} \, \mu\text{M} \cdot \text{1.s}^{-1}
$$

$$
K_{\text{off}} = 1 \times 10^{-4} \text{ s}^{-1}
$$

<figure><img src="image_3.png" /></figure>

B09: K<sub>D</sub>= 214 ± 8 nM

$$
K_{\text{on}} = 4.96 \times 10^{-4} \, \mu\text{M} \cdot \text{1.s}^{-1}
$$

$$
K_{\text{off}} = 1.06 \times 10^{-4} \text{ s}^{-1}
$$

<figure><img src="image_4.png" /></figure>

B10: K<sub>D</sub>= 21 ± 4 nM

$$
K_{\text{on}} = 1.91 \times 10^{-2} \, \mu\text{M} \cdot \text{1.s}^{-1}
$$

$$
K_{\text{off}} = 4.11 \times 10^{-4} \text{ s}^{-1}
$$

<figure><img src="image_5.png" /></figure>

VHH1-B07

<figure><img src="image_6.png" /></figure>

VHH1-B09

<figure><img src="image_7.png" /></figure>

VHH1-B10

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Fig. 2</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 3</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Fig. 4</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<table><thead><tr><th>VHH</th><th>Delta</th><th>BA.1</th><th>BQ.1.1</th><th>XBB.1.5</th><th>XBB.1.16.1</th><th>EG.5.1.3</th><th>BA.2.86.1</th></tr></thead><tbody><tr><td>B07</td><td>29.53</td><td>14.22</td><td>5.18</td><td>9.00</td><td>5.44</td><td>3.20</td><td>1.45</td></tr><tr><td>B09</td><td>18.02</td><td>27.11</td><td>7.77</td><td>8.29</td><td>11.77</td><td>7.75</td><td>3.35</td></tr><tr><td>B10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Ctl</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

IC50 (µg/mL)
<figure><img src="image_18.png" /><figcaption>Fig. 5</figcaption></figure>
<caption>Fig. 6</caption>

B07-Fc: $K_D^{App} = 0.11 \pm 0.0006$ nM
$K_{on} = 1.950$ µM·s⁻¹
$K_{off} = 2.14 \times 10^{-4}$ s⁻¹

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<table><thead><tr><th>IC50</th><th>Delta</th><th>BA.1</th><th>BQ.1.1</th><th>XBB.1</th><th>XBB.1.16</th><th>EG.5.1</th><th>BA.2.86</th><th>JN.1.1</th><th>KP.3.3</th></tr></thead><tbody><tr><td>mg/mL</td><td>0.23</td><td>0.19</td><td>0.11</td><td>0.12</td><td>0.09</td><td>0.08</td><td>0.10</td><td>0.13</td><td>0.33</td></tr><tr><td>nM</td><td>2.86</td><td>2.32</td><td>1.42</td><td>1.46</td><td>1.19</td><td>0.96</td><td>1.26</td><td>1.68</td><td>4.15</td></tr></tbody></table>

<table><thead><tr><th>IC50</th><th>JN.1.1</th><th>KP.3.3</th></tr></thead><tbody><tr><td>µg/mL</td><td>4.55x10<sup>-3</sup></td><td>5.98x10<sup>-3</sup></td></tr><tr><td>nM</td><td>0.06</td><td>0.07</td></tr></tbody></table>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

### Hamster ACE2

B07-Fc: $K_D^{App} = 78 \pm 1$ nM

$K_{on} = 3 \times 10^{-3}$ µM·s⁻¹

$K_{off} = 2.31 \times 10^{-4}$ s⁻¹

[B07-Fc]

600 nM

150 nM

37 nM

18 nM

9 nM
a

<figure><img src="image_30.png" /></figure>

b

<figure><img src="image_31.png" /></figure>

c

<figure><img src="image_32.png" /></figure>

d

<figure><img src="image_33.png" /></figure>

e

<figure><img src="image_34.png" /></figure>

f

<figure><img src="image_35.png" /></figure>

g

<figure><img src="image_36.png" /></figure>

h

<figure><img src="image_37.png" /></figure>
Supplementary Figures

Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2
binding and protects against infection *in vivo*

Simon Blachier¹, Marie-Christine Vaney², Laurine Conquet³†, Isabelle Staropoli⁴†, Ignacio Fernández², Emilie Giraud⁵, Atousa Arbabian², Vincent Michel⁶, Fruzsina Szilagyi¹, Salomé Guez⁵, Alix Boucharlat⁵, Jeanne Chiaravalli⁵, Jaouen Tran-Rajau⁵, Evelyne Dufour⁷, Ahmed Haouz⁸, Stéphane Petres⁷, Delphine Planas⁴, Xavier Montagutelli³, Fabrice Agou⁵, Pierre Lafaye⁹, Gabriel Ayme⁹†, Olivier Schwartz⁴,¹⁰†, Felix A. Rey²†, Jost Enninga¹†, Anne Brelot¹*
Supplementary Figures

Figures

1. Sequence alignment of B07, B09, and B10 VHHs.

2. VHH binding on sACE2 and schematic of the binding competition assay.

3. VHHs binding on cells expressing ACE2

4. Interactions between hACE2 and VHH B10.

5. Sequence alignment of human, hamster and mouse ACE2.

6. Interactions of hACE2 with SARS-CoV-2 RBD (PDB: 6MOJ) and footprints of interactions in selected complexes.

7. Impact of hACE2 substitution on VHH binding

8. B07-Fc activities

9. Relative cell surface expression of ACE2 mutants.

10. Separate color (top) or black and white (bottom) images of Fig. 7d.

11. Effect of VHH B07-Fc on hamster ACE2.

Tables

1. Statistical analyses and IC50 related to Fig. 4a and 6g (S-Fuse assay).

2. Crystallization conditions, data collection and refinement statistics

3. Van der Waals and polar interactions between hACE2 and VHH B10

4. Van der Waals and polar interactions between hACE2 and VHH B07

5. Van der Waals contacts between hACE2 and VHH B07 and between hACE2 and RBDs for a selection of viruses using hACE2 as a receptor.
<figure><img src="image_38.png" /><figcaption>Supplementary Figure 1</figcaption></figure>

**Supplementary Fig. 1: Sequence alignment of B07, B09, and B10 VHHs.** In dark blue background are the residues strictly conserved; in light blue background are the residues conserved for 2 sequences; in white background, residues which are not conserved. Lines above the sequences mark the CDRs in the IMGT definition (in bleu) and in the KABAT definition (in black). The sequence numbering, above and under the sequences, is according to IMGT definition and follow the numbering of the structures, respectively for B10 and B07. The contacts of B10 and B07 with hACE2 are reported under each residue as defined by the symbols in the inset. The somatic mutations are in red with germline residues in smaller font underneath. The secondary structure elements of B10 and B07 structures are indicated above each sequence. The green ovals indicate the amino acid difference between B07 and B09. J is the junction segment.
<figure><img src="image_39.png" /><figcaption>Supplementary Figure 2</figcaption></figure>

**Supplementary Fig. 2: VHH binding on sACE2 and schematic of the binding competition assay.** a Kinetic analysis of sACE2 on B07-His, B09-His, and B10-His VHHs by BioLayer Interferometry (BLI) using various concentrations of sACE2 (as indicated on the figure). Ni-NTA biosensors were used to immobilize VHH-His. K<sub>D</sub> of each VHHs is indicated. One representative experiment out of three. b Schematic representation of the binding competition assay. Following a baseline step, the sACE2-Fc (5 µg/mL) was immobilized onto the AHC biosensors. After a second baseline step, a first VHH was applied (VHH1; 5 µg/mL). The sensor was dipped in a mixture of VHH1 + the competitor VHH2 at the same concentration (5 µg/mL).
<figure><img src="image_40.png" /><figcaption>Supplementary Figure 3</figcaption></figure>

**Supplementary Fig. 3: VHHs binding on cells expressing ACE2.** a Example of FACS sequential gating strategy for detection of hACE2 on cell surface of HEK293-hACE2 cells by VHH or mAbs. b ACE2 cell surface detection by the commercial anti-ACE2 antibody AF933 (R&D system, 1 µg/mL). c Spike binding to hACE2-expressing cells. Cells were incubated with soluble spike (S) protein (Wuhan) (10 µg/mL) and stained with an anti-S antibody. d B07, B09, and B10 binding on A549-hACE2 (top) and Vero E6 (bottom) cells. Cells were incubated with the VHHs (10 µg/mL), stained with an anti-myc antibody and a AF488-conjugated anti-mouse antibody, before being analyzed by flow cytometry. Parental A549 cells or myc-tag VHH IgE were used as control. Data are mean ± SD of at least 3 independent experiments. e Fluorescence diagram overlays: B07, B09, B10 efficacy on different cell lines expressing exogenous (A549-hACE2) or endogenous non-human ACE2 (Vero E6). Background (blue) corresponds to the fluorescence intensity obtained on parental cells (A549) or using a VHH control (anti-IgE VHH) (Vero E6).
<figure><img src="image_41.png" /><figcaption>Supplementary Figure 4</figcaption></figure>

**Supplementary Fig. 4: Interactions between hACE2 and VHH B10.** a Structure of hACE2/B07/B10 complex. b Rotated view from (a) as indicated and open-book representation of complex hACE2 and VHH B10 (for clarity the VHH B07 was not displayed). The surface of contacts between hAEC2 and VHH B10 are displayed in white and contoured in green. The buried surfaces (BSA) are indicated under each surface. c Open-book interactions as in (b) with B10 colored complementarity determining regions (CDRs) in green (CDR1), blue (CDR2), and red (CDR3). d Detailed interaction between hACE2 and B10 (hydrogen bonds in black dashed lines). e Electrostatic potential mapped on the surface of the structure of hACE2 and B10. f Impact of hACE2 E160A substitution on B10 binding. Cells were transfected with myc tagged or untagged hACE2 E160A (and eGFP), incubated with an anti-myc antibody or B10 (10 µg/ml) and stained with a mouse anti-myc antibody and a AF647-conjugated anti-mouse antibody before being analyzed by flow cytometry. Data are mean ± SD of two independent experiments.
<figure><img src="image_42.png" /><figcaption>★ polar interactions hACE2-VHH B07
▼ VdW interactions hACE2-VHH B07
★ polar interactions hACE2-VHH B10
▼ VdW interactions hACE2-VHH B10</figcaption></figure>

red background : amino acid strictly conserved

yellow background : amino acid properties conserved (polar, nonpolar)

green background : numbered CYS residues forming disulfide bond

<caption>Supplementary Fig. 5: Sequence alignment of human, hamster, and mouse ACE2.</caption>
<figure><img src="image_43.png" /><figcaption>Supplementary Figure 6</figcaption></figure>

**Supplementary Fig. 6:** Interactions of hACE2 with SARS-CoV-2 RBD (PDB: 6M0J) and footprints of interactions in different complexes. a Structure of hACE2 (pink) / RBD SARS-CoV-2 (green) (PDB 6M0J) displayed as surfaces. b Open-book representation and footprints (in white) of RBD on hACE2 (left) and hACE2 on RBD (right). The buried surface (BSA) are indicated under each surface. The residues in hACE2 also involved in the interactions with VHH B07 are underlined. c Detailed interactions between hACE2 and RBD (hydrogen bonds in yellow dashed lines). d Footprints of VHH B07 and RBDs on ACE2 for a selection of viruses. Top, structures of complexes as indicated. Bottom, 90° views from top, in which the footprints (in white) of each molecule (VHH B07 and RBDs) on the top views are displayed on ACE2. e Each footprint is represented as an outline colored according to (d) and superposed on the hACE2/VHH B07/VHH B10 structure showing their overlap.
<figure><img src="image_44.png" /><figcaption>Supplementary Figure 7</figcaption></figure>

b

<figure><img src="image_45.png" /></figure>

**Supplementary Fig. 7: Impact of hACE2 substitution on VHH binding.** **a** Example of FACS sequential gating strategy for B07 detection of hACE2 at the cell surface of HEK293 cells co-transfected with plasmids coding hACE2 and eGFP (ratio 5/1, respectively). **b** Impact of alanine substitution on B09 binding. Cells were transfected with untagged or myc tag hACE2 alanine mutants (and eGFP), incubated with an anti-myc antibody or B09 (10 µg/mL) and stained with a mouse anti-myc antibody and a AF647-conjugated anti-mouse antibody before being analyzed by flow cytometry. Data are mean ± SD of at least three independent experiments.
Supplementary Figure 8

<figure><img src="image_46.png" /></figure>

**Supplementary Fig. 8: B07-Fc activities.** a B07-Fc binding on A549-hACE2 and Vero E6 cells. Cells were incubated with 0.1 µg/mL B07-Fc and a AF488-conjugated secondary antibody, and analyzed as in Fig 6b. Data are mean ± SD of at least 3 independent experiments. b Fluorescence diagram overlays performed as in Fig. 6c on A549-hACE2 and Vero E6 cells. c B07-Fc detection of hACE2 on ciliated cells of human nasal epithelial cells (hNEC). Representative immunofluorescence staining of hACE2 (Red: B07-Fc staining) in combination with α-tubulin (Green). Scale bars: left, 10 µm; right, 5 µm. d Vero E6 cells viability in the presence of B07-Fc measured by quantitating ATP using the CellTiter-Glo assay (triplicates). e Inhibition of SARS-CoV-2 replication by B07-Fc measured by RT-qPCR. Vero E6 cells pre-incubated with B07-Fc were infected with Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5 and XBB.1.16.1 (MOI and incubation time adjusted depending on virus replication, see Methods) and viral replication was measured by quantitative RT-qPCR (one experiment in triplicates).
<figure><img src="image_47.png" /><figcaption>Supplementary Figure 9</figcaption></figure>

<figure><img src="image_48.png" /></figure>

**Supplementary Fig. 9: Relative cell surface expression of ACE2 mutants and impact of substitutions on B07-Fc binding.** HEK293 cells transfected with plasmids encoding myc-hACE2-WT or the indicated mutant were stained with an antibody against the myc epitope or with B07-Fc and analyzed by flow cytometry. **a** Bars represent staining efficiency for cells expressing myc-hACE2 mutants relative to cells expressing myc-hACE2-WT after anti-myc staining. Data are means ± SD of at least three experiments. **b** Alignment of the amino acid sequences of human, hamster, and mouse ACE2 from position 27 to 42. Residues involved in B07 epitope are in bold. Purple stars indicate polar interactions between hACE2 and B07. **c** Bars represent HEK293 cells transfected with plasmids encoding myc-hACE2 (top) or myc-hamster ACE2 (bottom) and stained with an antibody against the myc epitope (left) or with B07-Fc (right) and analyzed by flow cytometry.
<figure><img src="image_49.png" /><figcaption>Supplementary Figure 10</figcaption></figure>

**Supplementary Fig. 10: Separate color (top) or black and white (bottom) images of Fig. 7d.** Phalloidin (F-actin), SARS-CoV-2 nucleocapsid (N) (viral replication), and tubulin staining on nasal respiratory epithelium, olfactory epithelium and lung bronchioli extracted from K18-hACE2 uninfected mice, and animals that received VHH-Fc (Ctl-Fc or B07-Fc). Representative immunofluorescence staining of phalloidin (Cyan), SARS-CoV-2 N protein (Red), and tubulin (Green). Scale bars: 10 µm.
a

<figure><img src="image_50.png" /></figure>

b

<figure><img src="image_51.png" /></figure>

**Supplementary Fig. 11: Effect of VHH B07-Fc on hamster.** a Schematic diagram showing the experimental design of B07-Fc prophylaxis in XBB.1.16.1 infected hamsters. Animals received intranasally (i.n.) 5 mg/kg VHH-B07-Fc (B07-Fc) or 5 mg/kg VHH-Fcctl (ctl-Fc). Twenty four hours later, they were infected with 10⁴ PFU XBB.1.16.1 intranasally (i.n.). Three days post-infection, lungs were collected for analysis. Animal behavior and weight were followed each day. b RNA load measured by RT-qPCR of SARS-CoV-2 in lung. Data are mean ± SD of 4 animals. Mann-Witney test: P value 0.0286*.
Supplementary Table 1

a

<table><thead><tr><th>B07</th><th>P values CI (µg/mL)</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>Delta</td><td>16.97 to 51.41</td><td>0.8712</td></tr><tr><td>BA.1</td><td>8.563 to 23.63</td><td>0.8616</td></tr><tr><td>BQ.1.1</td><td>3.885 to 6.914</td><td>0.9890</td></tr><tr><td>XBB1.5</td><td>6.116 to 13.25</td><td>0.9192</td></tr><tr><td>XBB.1.16.1</td><td>4.261 to 6.938</td><td>0.9909</td></tr><tr><td>EG.5.1.3</td><td>2.628 to 3.891</td><td>0.9948</td></tr><tr><td>BA.2.86.1</td><td>0.7223 to 2.903</td><td>0.9412</td></tr></tbody></table>

<table><thead><tr><th>B09</th><th>P values CI (µg/mL)</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>Delta</td><td>12.39 to 26.22</td><td>0.8901</td></tr><tr><td>BA.1</td><td>17.52 to 41.94</td><td>0.9223</td></tr><tr><td>BQ.1.1</td><td>4.839 to 12.49</td><td>0.9711</td></tr><tr><td>XBB1.5</td><td>6.435 to 10.67</td><td>0.9669</td></tr><tr><td>XBB.1.16.1</td><td>8.562 to 16.19</td><td>0.9815</td></tr><tr><td>EG.5.1.3</td><td>4.643 to 12.95</td><td>0.9631</td></tr><tr><td>BA.2.86.1</td><td>1.300 to 8.622</td><td>0.8942</td></tr></tbody></table>

b

<table><thead><tr><th>VHH</th><th>Delta</th><th>BA.1</th><th>BQ.1.1</th><th>XBB.1.5</th><th>XBB.1.16.1</th><th>EG.5.1.3</th><th>BA.2.86.1</th></tr></thead><tbody><tr><td>B07</td><td>1937.2</td><td>932.8</td><td>339.8</td><td>590.4</td><td>356.9</td><td>209.9</td><td>95.1</td></tr><tr><td>B09</td><td>1180.7</td><td>1776.3</td><td>509.1</td><td>543.2</td><td>771.2</td><td>507.8</td><td>219.5</td></tr><tr><td>B10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>CtI</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

IC50 (nM)

c

<table><thead><tr><th>S-Fuse</th><th>p-values CI (µg/mL)</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>Delta</td><td>0.1714 to 0.3072</td><td>0.9702</td></tr><tr><td>BA.1</td><td>0.1603 to 0.2168</td><td>0.9891</td></tr><tr><td>BQ.1.1</td><td>0.09368 to 0.1397</td><td>0.9761</td></tr><tr><td>XBB1.5</td><td>0.07860 to 0.1756</td><td>0.9420</td></tr><tr><td>XBB.1.16.1</td><td>0.08340 to 0.1088</td><td>0.9908</td></tr><tr><td>EG.5.1.3</td><td>0.06759 to 0.08863</td><td>0.9923</td></tr><tr><td>BA.2.86.1</td><td>0.07032 to 0.1454</td><td>0.9546</td></tr><tr><td>JN.1.1</td><td>0.1222 to 0.1561</td><td>0.9862</td></tr><tr><td>KP3.3</td><td>0.2897 to 0.3875</td><td>0.9801</td></tr></tbody></table>

<table><thead><tr><th>Anti-N</th><th>p-values CI (ng/mL)</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>JN.1.1</td><td>3.71 to 5.50</td><td>0.9928</td></tr><tr><td>KP.3.3</td><td>5.45 to 6.56</td><td>0.9966</td></tr></tbody></table>

**Supplementary Table 1: Statistical analyses and IC50 related to Fig. 4a and 6g (S-Fuse assay) and 6h (N detection).**

a P-values 95% confidence intervals for VHH IC50 values and R² values relative to B07 and B09 IC50s (Fig. 4a). b B07 IC50 values in nM (Fig. 4a). c P-values 95% confidence intervals and R² values relative to B07-Fc IC50 obtained in S-Fuse (Fig. 6g) or inhibition infection (anti-N) assays (Fig. 6h).
Supplementary Table 2

<table><thead><tr><th>Proteins</th><th>hACE2 - VHH B07 - VHH B10</th></tr></thead><tbody><tr><td><strong>Crystallization conditions</strong></td><td></td></tr><tr><td>Concentration (mg/mL)</td><td>7.9</td></tr><tr><td>Temperature (°C)</td><td>18</td></tr><tr><td>Crystallization buffer</td><td>0.1M HEPES pH 7.5<br>10% (w/v) 2-propanol, 20% (w/v) PEG 4K</td></tr><tr><td>Cryo-protectant</td><td>20% (v/v) Glycerol</td></tr><tr><td><strong>Data collection<sup>£</sup></strong></td><td></td></tr><tr><td>Synchrotron/Beamline</td><td>SOLEIL / PROXIMA-2A</td></tr><tr><td>Detector</td><td>EIGER X 9M</td></tr><tr><td>Space group</td><td>P 1 2<sub>1</sub> 1</td></tr><tr><td><strong>Unit cell parameters</strong></td><td></td></tr><tr><td>a,b,c (Å)</td><td>97.9, 108.1, 146.1</td></tr><tr><td>α, β, γ (°)</td><td>90, 98.9, 90</td></tr><tr><td>High resolution (Å) cut-off at CC<sub>1/2</sub>>30%</td><td>2.69</td></tr><tr><td>Resolution (Å)</td><td>48.37-2.69 (2.76-2.69)</td></tr><tr><td>Rmerge (%)</td><td>23.6 (159)</td></tr><tr><td>Rmeas (%)</td><td>25.5 (171)</td></tr><tr><td>Rpim (%)</td><td>9.5 (63.6)</td></tr><tr><td>Measured reflections</td><td>590 491 (43 028)</td></tr><tr><td>Unique reflections</td><td>83 613 (6 015)</td></tr><tr><td>&lt;l/σl&gt;</td><td>9.2 (1.3)</td></tr><tr><td>Completeness (%)</td><td>99.8 (97)</td></tr><tr><td>Multiplicity</td><td>7.1 (7.2)</td></tr><tr><td>&lt;CC<sub>1/2</sub>&gt; (%)</td><td>99.2 (37.5)</td></tr><tr><td>B-Wilson (Å<sup>2</sup>)</td><td>63.8</td></tr><tr><td>Number of molecules/au</td><td>3x(ACE2 - VHH B07 - VHH B10)</td></tr><tr><td><strong>Refinement<sup>£</sup></strong></td><td></td></tr><tr><td>Refinement program</td><td>BUSTER/TNT</td></tr><tr><td>Resolution</td><td>48.3-2.69 (2.71-2.69)</td></tr><tr><td>Number of reflections Work/Free</td><td>83 594 (1589) / 4 180 (83)</td></tr><tr><td>Rwork (%) / Rfree (%)</td><td>21.9 (34.1) / 25.4 (39.1)</td></tr><tr><td><strong>Root mean square deviation</strong></td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.007</td></tr><tr><td>Bond angles (°)</td><td>0.87</td></tr><tr><td><strong>Number of atoms</strong></td><td></td></tr><tr><td>Protein</td><td>20 123</td></tr><tr><td>Solvent</td><td>104</td></tr><tr><td>Chloride ions</td><td>4</td></tr><tr><td>Glycans</td><td>13</td></tr><tr><td>B-mean (Å<sup>2</sup>) (all atoms)</td><td>70.2</td></tr><tr><td><strong>Ramachandran (%)<sup>§</sup></strong></td><td></td></tr><tr><td>Favoured / Allowed / Outliers (%)</td><td>97 / 2.88 / 0.12</td></tr></tbody></table>

PEG, polyethylene glycol

£ Highest-resolution shell statistics is shown in parenthesis; au, asymmetric unit

Rmerge = ZhΣi(|<Ih> - Ih,i|)/ZhΣi Ih,i (where h are the unique reflections and i their symmetry-equivalent)

<l/σ(l)>, mean empirical signal-to-noise ratioCC(1/2), mean half-set correlation coefficient

Rwork = Σ|Fo - Fc|/Σ|Fo|; Rfree = Σ|Fo - Fc|/Σ|Fo| using 5% of the Fo selected randomly

§ Ramachandran statistics were calculated with MolProbity
Supplementary Table 3

Residues in hACE2 and VHH B10 involved in van der Waals contacts

<table><thead><tr><th>hACE2</th><th>ss</th><th>H/S</th><th>ASA (Å<sup>2</sup>)</th><th>BSA (Å<sup>2</sup>)</th><th>ΔiG (kcal/mol)</th></tr></thead><tbody><tr><td>VAL 132</td><td>β1</td><td></td><td>1.74</td><td>1.23</td><td>-0.01</td></tr><tr><td>CYS 133</td><td>β1</td><td></td><td>37.99</td><td>1.28</td><td>0.02</td></tr><tr><td>ASN 134</td><td>β1</td><td></td><td>59.59</td><td>27.51</td><td>-0.23</td></tr><tr><td>ASN 137</td><td>β2</td><td>H</td><td>61.75</td><td>16.45</td><td>-0.28</td></tr><tr><td>GLU 140</td><td>β2</td><td>H</td><td>103.32</td><td>79.65</td><td>-0.41</td></tr><tr><td>CYS 141</td><td>β2</td><td></td><td>37.18</td><td>3.08</td><td>0.05</td></tr><tr><td>LEU 142</td><td>β2</td><td></td><td>57.57</td><td>32.86</td><td>0.52</td></tr><tr><td>PRO 146</td><td>loop<br/>β2-α5</td><td></td><td>83.77</td><td>2.68</td><td>0.04</td></tr><tr><td>GLU 150</td><td>α5</td><td>H</td><td>111.20</td><td>74.53</td><td>-0.51</td></tr><tr><td>ILE 151</td><td>α5</td><td></td><td>25.26</td><td>24.42</td><td>0.39</td></tr><tr><td>ASN 154</td><td>α5</td><td></td><td>110.53</td><td>43.86</td><td>0.29</td></tr><tr><td>SER 155</td><td>loop<br/>α5-α6</td><td></td><td>14.63</td><td>12.32</td><td>0.15</td></tr><tr><td>LEU 156</td><td>loop<br/>α5-α6</td><td></td><td>122.74</td><td>70.82</td><td>1.01</td></tr><tr><td>ASP 157</td><td>loop<br/>α5-α6</td><td>HS</td><td>71.03</td><td>54.04</td><td>0.09</td></tr><tr><td>ASN 159</td><td>α6</td><td>H</td><td>89.83</td><td>23.87</td><td>0.24</td></tr><tr><td>GLU 160</td><td>α6</td><td>H</td><td>75.85</td><td>75.85</td><td>-0.26</td></tr><tr><td>TRP 163</td><td>α6</td><td></td><td>107.99</td><td>51.76</td><td>0.81</td></tr><tr><td>VAL 298</td><td>α11</td><td></td><td>93.84</td><td>4.66</td><td>0.07</td></tr></tbody></table>

<table><thead><tr><th>VHH B10</th><th>CDR</th><th>H/S</th><th>ASA (Å<sup>2</sup>)</th><th>BSA (Å<sup>2</sup>)</th><th>ΔiG (kcal/mol)</th></tr></thead><tbody><tr><td>THR 28</td><td>FR1</td><td></td><td>72.00</td><td>6.86</td><td>0.11</td></tr><tr><td>SER 30</td><td>FR1</td><td></td><td>55.90</td><td>1.47</td><td>-0.02</td></tr><tr><td>SER 31</td><td>CDR1</td><td></td><td>53.24</td><td>34.54</td><td>0.28</td></tr><tr><td>TYR 32</td><td>CDR1</td><td></td><td>55.51</td><td>23.51</td><td>0.04</td></tr><tr><td>SER 52</td><td>CDR2</td><td></td><td>4.26</td><td>2.25</td><td>0.03</td></tr><tr><td>SER 54</td><td>CDR2</td><td>H</td><td>54.21</td><td>10.68</td><td>0.08</td></tr><tr><td>THR 56</td><td>CDR2</td><td>H</td><td>125.88</td><td>16.31</td><td>-0.14</td></tr><tr><td>SER 57</td><td>CDR2</td><td></td><td>48.79</td><td>10.31</td><td>-0.00</td></tr><tr><td>TYR 59</td><td>CDR2</td><td>H</td><td>98.43</td><td>39.18</td><td>0.28</td></tr><tr><td>ALA 75</td><td>FR3</td><td></td><td>98.79</td><td>4.67</td><td>0.07</td></tr><tr><td>LYS 100</td><td>CDR3</td><td></td><td>92.82</td><td>28.85</td><td>0.07</td></tr><tr><td>GLY 101</td><td>CDR3</td><td></td><td>21.23</td><td>21.06</td><td>0.34</td></tr><tr><td>PHE 102</td><td>CDR3</td><td>H</td><td>98.61</td><td>95.00</td><td>1.38</td></tr><tr><td>SER 103</td><td>CDR3</td><td>H</td><td>36.15</td><td>29.79</td><td>0.33</td></tr><tr><td>THR 104</td><td>CDR3</td><td></td><td>14.96</td><td>4.54</td><td>-0.05</td></tr><tr><td>ILE 105</td><td>CDR3</td><td></td><td>151.17</td><td>138.96</td><td>1.99</td></tr><tr><td>TYR 106</td><td>CDR3</td><td>H</td><td>151.51</td><td>89.10</td><td>0.91</td></tr><tr><td>ASN 107</td><td>CDR3</td><td></td><td>0.62</td><td>0.62</td><td>-0.01</td></tr><tr><td>GLN 108</td><td>CDR3</td><td>H</td><td>58.04</td><td>16.87</td><td>-0.28</td></tr><tr><td>ARG 112</td><td>CDR3</td><td>HS</td><td>54.59</td><td>26.41</td><td>-0.67</td></tr></tbody></table>

Polar contacts between hACE2 and VHH B10

<table><thead><tr><th colspan="4">hACE2</th><th colspan="2">VHH B10</th></tr><tr><th>atom</th><th>ss</th><th colspan="2">distance (Å)</th><th>atom</th><th>CDR</th></tr></thead><tbody><tr><td>ASN 137 OD1</td><td>β2</td><td>H</td><td>3.61</td><td>TYR 59 OH</td><td>CDR2</td></tr><tr><td>GLU 140 OE2</td><td>β2</td><td>H</td><td>3.40</td><td>GLN 108 NE2</td><td>CDR3</td></tr><tr><td>GLU 150 OE2</td><td>α5</td><td>H</td><td>2.83</td><td>SER 54 OG</td><td>CDR2</td></tr><tr><td>GLU 150 OE2</td><td>α5</td><td>H</td><td>3.36</td><td>THR 56 OG1</td><td>CDR2</td></tr><tr><td>ASP 157 OD2</td><td>α6</td><td>HS</td><td>3.63</td><td>ARG 112 NH1</td><td>FR4</td></tr><tr><td>ASP 157 OD2</td><td>α6</td><td>HS</td><td>2.93</td><td>ARG 112 NH2</td><td>FR4</td></tr><tr><td>ASN 159 O</td><td>α6</td><td>H</td><td>3.57</td><td>TYR 106 OH</td><td>CDR2</td></tr><tr><td>GLU 160 OE1</td><td>α6</td><td>H</td><td>2.58</td><td>SER 103 OG</td><td>CDR3</td></tr><tr><td>GLU 160 OE1</td><td>α6</td><td>H</td><td>3.10</td><td>SER 103 N</td><td>CDR3</td></tr><tr><td>GLU 160 OE2</td><td>α6</td><td>H</td><td>2.88</td><td>PHE 102 N</td><td>CDR2</td></tr></tbody></table>

**Supplementary Table 3: Van der Waals and polar interactions between hACE2 and VHH B10.** CDR: complementary-determining region (Kabat definition); FR: Framework region; ss: secondary structure in hACE2; H: hydrogen bonds; S: salt-bridges; ASA: accessible surface area; BSA: buried surface area; ΔGi: solvation energy effect; Vertical bars in BSA columns indicate the buried area percentage (with one bar per 10% of ASA).
Supplementary Table 4

Residues in hACE2 and VHH B07 involved in van der Waals contacts

<table><thead><tr><th>hACE2</th><th>ss</th><th>HS</th><th>ASA (Å<sup>2</sup>)</th><th>BSA (Å<sup>2</sup>)</th><th>ΔiG (kcal/mol)</th></tr></thead><tbody><tr><td>THR 27</td><td>α1</td><td></td><td>71.69</td><td>13.25 ||</td><td>0.20</td></tr><tr><td>ASP 30</td><td>α1</td><td></td><td>99.65</td><td>18.60 ||</td><td>0.23</td></tr><tr><td>LYS 31</td><td>α1</td><td></td><td>121.90</td><td>48.19 ||||</td><td>0.77</td></tr><tr><td>HIS 34</td><td>α1</td><td>H</td><td>139.56</td><td>120.57 ||||||||</td><td>0.60</td></tr><tr><td>GLU 35</td><td>α1</td><td>HS</td><td>85.73</td><td>79.49 ||||||||</td><td>-0.32</td></tr><tr><td>GLU 37</td><td>α1</td><td>H</td><td>81.81</td><td>46.14 ||||</td><td>-0.54</td></tr><tr><td>ASP 38</td><td>α1</td><td>H</td><td>80.63</td><td>77.51 ||||||||</td><td>-0.25</td></tr><tr><td>LEU 39</td><td>α1</td><td></td><td>44.52</td><td>33.13 ||||||</td><td>0.53</td></tr><tr><td>GLN 42</td><td>α1</td><td></td><td>94.45</td><td>48.95 ||||</td><td>-0.11</td></tr><tr><td>LYS 68</td><td>α2</td><td>H</td><td>121.21</td><td>51.24 ||||</td><td>-0.91</td></tr><tr><td>ALA 71</td><td>α2</td><td></td><td>62.37</td><td>25.40 ||||</td><td>0.31</td></tr><tr><td>PHE 72</td><td>α2</td><td></td><td>14.52</td><td>14.52 ||||||||</td><td>0.21</td></tr><tr><td>GLU 75</td><td>α2</td><td>HS</td><td>94.37</td><td>54.58 ||||</td><td>-0.29</td></tr><tr><td>GLN 76</td><td>α2</td><td></td><td>8.80</td><td>2.04 ||</td><td>-0.02</td></tr><tr><td>LYS 353</td><td>loop<br>β4-β5</td><td>H</td><td>119.29</td><td>28.47 ||||</td><td>-0.85</td></tr></tbody></table>

<table><thead><tr><th>VHH B07</th><th>CDR</th><th>HS</th><th>ASA (Å<sup>2</sup>)</th><th>BSA (Å<sup>2</sup>)</th><th>ΔiG (kcal/mol)</th></tr></thead><tbody><tr><td>SER 30</td><td>FR1</td><td></td><td>58.84</td><td>1.96 ||</td><td>-0.02</td></tr><tr><td>THR 31</td><td>CDR1</td><td></td><td>101.13</td><td>52.93 ||||</td><td>0.21</td></tr><tr><td>ASN 32</td><td>CDR1</td><td>H</td><td>40.87</td><td>21.48 ||||</td><td>-0.26</td></tr><tr><td>THR 33</td><td>CDR1</td><td>H</td><td>50.34</td><td>47.92 ||||||</td><td>-0.09</td></tr><tr><td>TYR 37</td><td>FR2</td><td></td><td>8.16</td><td>1.11 ||</td><td>-0.01</td></tr><tr><td>LEU 47</td><td>FR2</td><td></td><td>55.19</td><td>25.76 ||||</td><td>0.41</td></tr><tr><td>SER 50</td><td>CDR2</td><td></td><td>17.02</td><td>16.04 ||||||||</td><td>0.07</td></tr><tr><td>ILE 51</td><td>CDR2</td><td></td><td>11.20</td><td>0.33 ||</td><td>0.01</td></tr><tr><td>SER 52</td><td>CDR2</td><td></td><td>29.38</td><td>29.38 ||||||</td><td>0.43</td></tr><tr><td>THR 53</td><td>CDR2</td><td>H</td><td>62.84</td><td>34.94 ||||</td><td>-0.20</td></tr><tr><td>SER 54</td><td>CDR2</td><td>H</td><td>93.93</td><td>22.99 ||</td><td>0.24</td></tr><tr><td>ASN 56</td><td>CDR2</td><td>H</td><td>112.21</td><td>21.83 ||</td><td>0.03</td></tr><tr><td>THR 57</td><td>CDR2</td><td></td><td>74.73</td><td>3.97 ||</td><td>0.01</td></tr><tr><td>TYR 58</td><td>CDR2</td><td></td><td>127.07</td><td>101.47 ||||||</td><td>0.73</td></tr><tr><td>PRO 60</td><td>CDR2</td><td></td><td>26.45</td><td>1.84 ||</td><td>0.03</td></tr><tr><td>PRO 98</td><td>CDR3</td><td></td><td>43.13</td><td>39.61 ||||||</td><td>0.63</td></tr><tr><td>SER 99</td><td>CDR3</td><td></td><td>71.98</td><td>55.05 ||||</td><td>-0.16</td></tr><tr><td>ARG 101</td><td>CDR3</td><td>HS</td><td>84.80</td><td>53.72 ||||</td><td>-1.48</td></tr><tr><td>ILE 102</td><td>CDR3</td><td></td><td>138.09</td><td>84.75 ||||</td><td>1.36</td></tr></tbody></table>

Polar contacts between hACE2 and VHH B07

<table><thead><tr><th colspan="4">hACE2</th><th colspan="2">VHH B07</th></tr><tr><th>atom</th><th>ss</th><th colspan="2">distance (Å)</th><th>atom</th><th>CDR</th></tr></thead><tbody><tr><td>HIS 34 NE2</td><td>α1</td><td>H</td><td>3.12</td><td>ASN 56 O</td><td>CDR2</td></tr><tr><td>GLU 35 OE1</td><td>α1</td><td>HS</td><td>3.01</td><td>ARG 101 NH1</td><td>CDR3</td></tr><tr><td>GLU 35 OE2</td><td>α1</td><td>HS</td><td>2.90</td><td>ARG 101 NH2</td><td>CDR3</td></tr><tr><td>GLU 37 OE1</td><td>α1</td><td>H</td><td>3.75</td><td>ASN 56 ND2</td><td>CDR2</td></tr><tr><td>GLU 37 OE1</td><td>α1</td><td>H</td><td>3.40</td><td>SER 54 OG</td><td>CDR2</td></tr><tr><td>ASP 38 OD1</td><td>α1</td><td>H</td><td>2.76</td><td>THR 53 OG1</td><td>CDR2</td></tr><tr><td>ASP 38 OD2</td><td>α1</td><td>H</td><td>2.79</td><td>THR 33 OG1</td><td>CDR1</td></tr><tr><td>ASP 38 OD2</td><td>α1</td><td>H</td><td>3.05</td><td>THR 53 N</td><td>CDR2</td></tr><tr><td>LYS 68 NZ</td><td>α2</td><td>H</td><td>3.26</td><td>ASN 32 OD1</td><td>CDR1</td></tr><tr><td>GLU 75 OE1</td><td>α2</td><td>HS</td><td>3.07</td><td>ARG 101 NE</td><td>CDR3</td></tr><tr><td>GLU 75 OE2</td><td>α2</td><td>HS</td><td>2.89</td><td>ARG 101 NH1</td><td>CDR3</td></tr><tr><td>LYS 353 NZ</td><td>loop<br>β4-β5</td><td>H</td><td>2.79</td><td>THR 53 OG1</td><td>CDR2</td></tr></tbody></table>

**Supplementary Table 4: Van der Waals and polar interactions between hACE2 and VHH B07.** CDR: complementary-determining region (Kabat definition); FR: Framework region; ss: secondary structure in hACE2; H: hydrogen bonds; S: salt-bridges; ASA: accessible surface area; BSA: buried surface area; ΔGi: solvation energy effect; Vertical bars in BSA columns indicate the buried area percentage (with one bar per 10% of ASA).
Supplementary Table 5

<table><thead><tr><th colspan="2">hACE2</th><th colspan="2">hACE2/VHH B07-B10 (this work)</th><th colspan="2">hACE2/RBD SARS CoV-2 (PDB 6M0J)</th><th colspan="2">hACE2/RBD SARS CoV (PDB 2AJF)</th><th colspan="2">hACE2/RBD HCoV-NL63 (PDB 3KBH)</th></tr><tr><th>residues</th><th>ss</th><th>HS</th><th>BSA (Å²) VHH B07</th><th>HS</th><th>BSA (Å²) RBD</th><th>HS</th><th>BSA (Å²) RBD</th><th>HS</th><th>BSA (Å²) RBD</th></tr></thead><tbody><tr><td>SER 19</td><td>N-ter</td><td></td><td></td><td></td><td>10.86 ||</td><td></td><td>31.48 ||</td><td></td><td></td></tr><tr><td>GLU 23</td><td>α1</td><td></td><td></td><td></td><td></td><td></td><td>1.24 ||</td><td></td><td></td></tr><tr><td>GLN 24</td><td>α1</td><td></td><td></td><td>H</td><td>54.04 ||||||</td><td>H</td><td>39.35 ||||</td><td></td><td></td></tr><tr><td>THR 27</td><td>α1</td><td></td><td>13.25 ||</td><td></td><td>67.91 ||||||</td><td></td><td>73.41 ||||||</td><td></td><td></td></tr><tr><td>PHE 28</td><td>α1</td><td></td><td></td><td></td><td>14.77 ||||||</td><td></td><td>11.99 ||||||</td><td></td><td></td></tr><tr><td>ASP 30</td><td>α1</td><td></td><td>18.60 ||</td><td>HS</td><td>41.36 ||||</td><td></td><td>13.25 ||</td><td>22.50 ||</td><td></td></tr><tr><td>LYS 31</td><td>α1</td><td></td><td>48.19 ||||</td><td></td><td>91.32 ||||||</td><td></td><td>52.47 ||||||</td><td>3.56 ||</td><td></td></tr><tr><td>ASN 33</td><td>α1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>20.55 ||||||</td><td></td></tr><tr><td>HIS 34</td><td>α1</td><td>H</td><td>120.57 ||||||||</td><td>H</td><td>74.52 ||||</td><td></td><td>62.39 ||||</td><td>61.01 ||||</td><td></td></tr><tr><td>GLU 35</td><td>α1</td><td>HS</td><td>79.49 ||||||||</td><td>H</td><td>14.69 ||</td><td></td><td></td><td></td><td></td></tr><tr><td>GLU 37</td><td>α1</td><td>H</td><td>46.14 ||||</td><td>H</td><td>14.30 ||||</td><td>H</td><td>17.13 ||||</td><td>H</td><td>37.61 ||||||</td></tr><tr><td>ASP 38</td><td>α1</td><td>H</td><td>77.51 ||||||||</td><td>H</td><td>32.56 ||||</td><td>H</td><td>34.28 ||||</td><td></td><td>1.97 ||</td></tr><tr><td>LEU 39</td><td>α1</td><td></td><td>33.13 ||||||</td><td></td><td></td><td>H</td><td></td><td></td><td></td></tr><tr><td>TYR 41</td><td>α1</td><td></td><td></td><td>H</td><td>42.44 ||||||||</td><td>H</td><td>44.99 ||||||||</td><td>H</td><td>35.07 ||||||</td></tr><tr><td>GLN 42</td><td>α1</td><td></td><td>48.95 ||||</td><td>H</td><td>46.41 ||||</td><td>H</td><td>32.19 ||||</td><td></td><td></td></tr><tr><td>LEU 45</td><td>α1</td><td></td><td></td><td></td><td>27.84 ||||</td><td></td><td>33.41 ||||</td><td></td><td></td></tr><tr><td>LYS 68</td><td>α2</td><td>H</td><td>51.24 ||||</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALA 71</td><td>α2</td><td></td><td>25.40 ||||</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHE 72</td><td>α2</td><td></td><td>14.52 ||||||||</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GLU 75</td><td>α2</td><td>HS</td><td>54.58 ||||</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GLN 76</td><td>α2</td><td></td><td>2.04 ||||</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LEU 79</td><td>α2</td><td></td><td></td><td></td><td>25.95 ||||</td><td></td><td>31.12 ||||</td><td></td><td></td></tr><tr><td>MET 82</td><td>α2-η1</td><td></td><td></td><td></td><td>27.86 ||||</td><td></td><td>37.04 ||||</td><td></td><td></td></tr><tr><td>TYR 83</td><td>α2-η1</td><td></td><td></td><td>H</td><td>36.50 ||||||||</td><td>H</td><td>31.15 ||||||</td><td></td><td></td></tr><tr><td>PRO 321</td><td>α12-α13</td><td></td><td></td><td></td><td></td><td></td><td></td><td>19.46 ||||</td><td></td></tr><tr><td>ASN 322</td><td>α12-α13</td><td></td><td></td><td></td><td></td><td></td><td></td><td>12.31 ||</td><td></td></tr><tr><td>MET 323</td><td>α12-α13</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1.96 ||</td><td></td></tr><tr><td>THR 324</td><td>α12-α13</td><td></td><td></td><td></td><td>2.18 ||</td><td></td><td>5.02 ||</td><td>H</td><td>44.17 ||||||||</td></tr><tr><td>GLN 325</td><td>α13</td><td></td><td></td><td></td><td>8.02 ||</td><td>H</td><td>43.00 ||||</td><td></td><td>42.15 ||||</td></tr><tr><td>GLY 326</td><td>α13</td><td></td><td></td><td></td><td>2.01 ||</td><td></td><td>13.88 ||||</td><td></td><td>19.16 ||||||||</td></tr><tr><td>GLU 329</td><td>α13</td><td></td><td></td><td></td><td></td><td>HS</td><td>27.64 ||||</td><td></td><td></td></tr><tr><td>ASN 330</td><td>α13</td><td></td><td></td><td></td><td>28.75 ||||</td><td>H</td><td>33.10 ||||</td><td></td><td>15.67 ||</td></tr><tr><td>LYS 353</td><td>loop β4-β5</td><td>H</td><td>28.47 ||||</td><td>H</td><td>98.99 ||||||||</td><td>H</td><td>104.54 ||||||||</td><td>H</td><td>65.15 ||||||</td></tr><tr><td>GLY 354</td><td>β5</td><td></td><td></td><td></td><td>30.22 ||||</td><td></td><td>27.83 ||||</td><td></td><td>54.04 ||||||||</td></tr><tr><td>ASP 355</td><td>β5</td><td></td><td></td><td></td><td>12.29 ||||||||</td><td></td><td>10.29 ||||||||</td><td></td><td>15.47 ||||||||</td></tr><tr><td>PHE 356</td><td>β5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>10.01 ||||||||</td></tr><tr><td>ARG 357</td><td>β5</td><td></td><td></td><td></td><td>12.01 ||||||</td><td></td><td>10.82 ||||||</td><td></td><td>3.79 ||||</td></tr><tr><td>MET 383</td><td>α14</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>30.27 ||||||</td></tr><tr><td>ALA 384</td><td>α14</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1.90 ||</td></tr><tr><td>ALA 386</td><td>α14</td><td></td><td></td><td></td><td>0.25 ||</td><td></td><td></td><td></td><td>23.96 ||||||</td></tr><tr><td>ALA 387</td><td>α14</td><td></td><td></td><td></td><td></td><td></td><td></td><td>H</td><td>40.36 ||||</td></tr><tr><td>PRO 390</td><td>η7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1.57 ||</td></tr><tr><td>ARG 393</td><td>η7-α15</td><td></td><td></td><td>H</td><td>10.70 ||</td><td></td><td>6.70 ||</td><td></td><td>23.51 ||||</td></tr><tr><td>PHE 555</td><td>α22</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>10.92 ||</td></tr></tbody></table>

Supplementary Table 5: Van der Waals contacts between hACE2 and VHH B07 and between hACE2 and RBDs for a selection of viruses using hACE2 as a receptor. ss: secondary structure of hACE2; H: hydrogen bonds; S: salt-bridges; BSA: buried surface area; Vertical bars in BSA columns indicate the buried area percentage (with one bar per 10% of ASA of each indicated residue). In bold red are indicated the common interactions between B07 and SARS-CoV-2 RBD structures. In bold red italic are indicated the common interactions between the four structures.