Evolution of SARS-CoV-2 Spikes shapes their binding affinities to animal ACE2 orthologs

ABSTRACT Spike-receptor interaction is a critical determinant for the host range of coronaviruses. Here, we investigated all the five World Health Organization-designated variants of concern (VOC), including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), for their Spike receptor-binding domain (RBD)’s interactions with ACE2 orthologs of 18 animal species. We found that, compared to the RBD of an early isolate WHU01, the Alpha RBD has markedly increased affinity to cattle and pig ACE2 proteins and decreased affinity to horse and donkey ACE2 proteins. The RBDs of Beta and Gamma variants have almost completely lost affinity to bat, horse, and donkey ACE2 orthologs. Mainly due to the Q493R and N501Y mutations, the Omicron RBD showed markedly enhanced affinity to mouse ACE2. Molecular dynamic simulations further suggest that Omicron RBDs are optimal for electrostatic interactions with mouse ACE2. Interestingly, the Omicron RBD also showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of horse, donkey, pig, dog, cat, pangolin, American pika, and bat. The K417N, G496S, and Y505H substitutions were identified as three major contributors that commonly have negative impact on RBD binding to these eight ACE2 orthologs. These findings show that Spike mutations have been continuously shaping SARS-CoV-2’s binding affinities to animal ACE2 orthologs and suggest the importance of surveillance of animal infection by circulating SARS-CoV-2 variants. IMPORTANCE Spike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant.

1. Line 64-65, progresses have been achieved in developing prophylactic vaccines and antibody therapeutics (1-13).There are small molecule drugs like Paxlovid against SARS-COV-2, please revise and add references. 2. Line 74, according to Organization (WHO) online updates, need to include web link as citation.3. Line 82, SARS-CoV-2 utilizes ACE2 as an essential cellular receptor (26,32,33).Suggest adding sentences about RBD here, where it is located, its significance and how RBD interacts with ACE2. 4. How to define ACE2 high and low cells?<1000 MFI is low and >4000 as high? 5. Line 119-20, to more and more ACE2.What does this mean?revise.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin the revision process.The information that you entered when you first submitted the paper will be displayed.Please update the information as necessary.Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Response to Reviewers (reviewer comments in black; author responses in blue)
Reviewer #1 (Comments for the Author): Weitong et al investigated the binding affinities of spike protein to ACE2 orthologs of 18 animal species.This is a well-written paper with solid data support.The results in the paper help us understand the impact of spike mutations on ACE2 binding across mammalian species.
We appreciate the reviewer's careful evaluation and positive comments on the quality and significance of our study.
Major comments: 1.In Introduction, authors should briefly review surveillance studies of SARS-COV-2 in animal species, such as strains found in cats and dogs, zoo animals, and minks in farm.What animals can harbor SARS-COV-2 as carrier?Review previous studies of RBD-ACE2 binding in animals, and how this paper is distinct from other studies.
We thank the reviewer for these comments.As suggested, we have added to the Introduction section a brief review of the studies of SARS-COV-2 in animal species (lines 85-94).Regarding the comment -'review previous studies of RBD-ACE2 binding in animals, and how this paper is distinct from other studies', such a review had previously been included in the first paragraph of the Discussion section.We've kept the review and introduced a minor revision as shown in the revised main text lines 312-317: 'Similar studies involving a panel of animal ACE2 orthologs have previously only been performed for the prototype SARS-CoV-2 Spike (30,31,33,47).Since receptor usage is a critical determinant for the host range of coronaviruses (32), the current study concerning 108 distinct interactions (18 animal ACE2 orthologs × 6 major SARS-CoV-2 Spike variants) provides more comprehensive and useful information for understanding possible cross-species transmission risks of all the five VOCs'.Note that one of the previous studies of RBD-ACE2 binding in animals is our own study, which describes the interactions of 4 related CoVs (SARS-CoV-2, SARS-CoV-1, pangolin-CoV, bat-CoV RaTG13) with 15 animal ACE2 orthologs (Li et al.
2. How many replicates of measurements were performed for each data point in Figure 2C/D, is it possible some differences are due to experimental variation or batch effect?
We thank the reviewer for careful evaluation of our data.The experiments in Figure 2C/D were repeated twice with highly similar results.However, some very minor MFI differences might indeed come from experimental variation or batch difference, for example the MFI differences between the Delta/donkey (MFI: 2013 for ACE2-high, 213 for ACE2-low) vs Delta/goat (MFI: 2008 for ACE2-high, 225 for ACE2-low) interactions.Thus, in the Figures 3-7, we mainly focus on changes that are (1) more significant and (2) reproducible in both ACE2-high and ACE2-low cells in Figure 2. In addition, all the key findings from this flow cytometry analysis have then been further validated using surface plasmon resonance (SPR)-or Bio-layer interferometry (BLI)-based, more accurate, and quantitative binding kinetics studies (Fig 3B , 3E-H, 4B, 5B, 5G, 7A, 7C, S3D and S4B), mutagenesis studies (Fig 3E-H, 4C-H, 5E-H, 7B-D), and pseudovirus infection studies (Fig 3C , 4E, 4G, 5C, 5D, 5H, S6B).We believe that these multi-level validations offset the limitation/minor variations of the data in Figure 2C/D.This is also discussed in the Limitation of study section followed by the Discussion section.

3.
It seems all RBDs failed to bind rat ACE2 (no difference) compared to no RBD in ACE2 low cells, how to explain this (similar for civet)?
First, the finding in Figure 2C/D that WHU01 RBD fails to bind rat Ace2 is consistent with the rat-Ace2 binding data in our previous study  Coronaviruses Utilize Multiple ACE2 Orthologs and Are Potently Blocked by an Improved ACE2-Ig. J Virol. 2020. PMID: 32847856).Second, we can see significant binding of rat Ace2 to Alpha, Beta, and Gama RBDs in the ACE2-high cells (Fig 2C).Third, although the MFI signal of Alpha/Beta/Gama RBD binding to rat Ace2 looks weak in Figure 2D, the flow cytometry dot plot raw data in Figure S2 clearly show that Alpha, Beta, and Gama RBDs do bind rat Ace2.The following are high resolution images from Figure S2.The magenta boxes mark the RBD-positive cell populations in each sample.You can see rat Ace2 and RBD double positive cell populations in Alpha, Beta, and Gama RBD staining samples.Finally, these double-positive cell populations are so small in the rat Ace2-low cells that the binding can't be significantly reflected by MFI.This is likely because the interactions observed in ACE2-high cells are actually low-affinity interactions that rely on the avidity effect of high ACE2 density on the cell surface.Because of this reason, we think the interactions observed in the ACE2-low cells are more likely to be high-affinity interactions and thus more likely to be physiologically relevant.We therefore opt to focus more on the interactions or changes observed in the ACE2-low cells in the subsequent part of this study (Figs 3-7 ). Likely for the same reasons, interactions of civet Ace2 with multiple tested Spike variants are less significant/visible in ACE2-low cells (Fig 2D) than in ACE2-high cells (Fig 2C).

4.
Authors may explain better in Fig. 2 C/D, Fig 7B/D what implications of ACE2-high versus ACE2-low population data, are they equally important?How to explain the discordances between the two populations such as rat?
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred