Uncovering a conserved vulnerability site in SARS‐CoV‐2 by a human antibody

Abstract An essential step for SARS‐CoV‐2 infection is the attachment to the host cell receptor by its Spike receptor‐binding domain (RBD). Most of the existing RBD‐targeting neutralizing antibodies block the receptor‐binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non‐RBM‐targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a K D of 5.6 nM, neutralizes SARS‐CoV‐2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross‐reactivity against SARS‐CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted “ideal” vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20‐binding but confers little or no resistance to neutralization. Finally, in vitro mode‐of‐action characterization and negative‐stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS‐CoV‐2 Spike for the development of potential antiviral drugs.

1. Fig. 1A： For the screening, the authors mentioned that B cells from three convalescent patients were pooled. Are there any criteria for choosing the convalescent patients? At the end of the screening, how many neutralized antibodies were obtained besides scFD20? 2.Line 184 We next investigated the in vivo potential of FD20 by intraperitoneal injection 6 h before intranasal inoculation of hamsters by live viruses (Figure 3): Is this sentence meaning that FD20 was injected first and then virus infection after 6h? It looks strange since normally for antibody treatment, the animals should be infected with virus first and then inject antibody for treatment. 3. Fig. 3B and 3E：The virus titers in either the lung or the nasal tissues determined by PCR and TCID50 were not consistent. For example, there are two lung tissues showing 0 TCID50 (Fig. 3C) and the PCR results ( Fig. 3B) are quite high. What could be the reasons behind? It is quite hard to understand why the titers were varied so much between the PCR method and the TCID50 method.
Referee #2 (Remarks for Author): COVID-19, the pandemic caused by SARS-CoV-2, is still threatening thousands of millions of people around the world. In the absence of specific and highly effective medicine, the treatment of COVID-19 patient is very challenging, and neutralizing antibodies have great potential for treatment. There are many neutralization antibodies have been reported, most of them are targeting RBD, especially RBM, some are targeting NTD and S2. However, antibodies that directly against the RBM region may induce escape mutations. Therefore, more neutralization antibodies are still needed, especially antibodies that targeting conserved and functionally important regions on S protein, theoretically, which can reduce the occurrence of mutations and may exert a broad-spectrum antiviral effect against coronaviruses.
In this article, neutralizing antibody FD20 was isolated from COVID-19 convalescent patients through a phage display strategy. FD20 targets a conservative position and have a broad-spectrum antiviral effect. For characterization and functional study, the authors demonstrated FD20's high affinity to ACE2, the effectiveness on cell-based neutralization assays. In order to understand the mode-of-action, the authors resolved the co-crystal structure of FD20-S protein. Overall, the aim and main findings of this study are important, the design and logic are clear, the conclusions could be supported by the results. The manuscript is well-written. Thus, this reviewer endorses the publication of this work after revision.
Major points： 1. In most cases, the destination of a neutralization is for therapeutics. Thus, the in vivo (animal model) validation of its efficacy for protection or treatment is the key. Unfortunately, for animal model experiments, only marginal efficacy was observed. The authors need to improve the efficacy through practical ways, at least, the authors need to thoroughly discuss this. 2. The in vitro affinity of FD20 to ACE2 is very high, while the in vivo efficacy is very low. A reasonable explanation is necessary? Is it possible that the in vivo stability of FD20 is low? Or FD20 was eliminated rapidly through some yet-to-bediscovered mechanism? The reviewer suggests the authors to test the distribution of FD20 in animal model at different time points. 3. For kinetic measurement, to assure the reliability of the determined Kd, the range of the serial dilution should cover the Kd. However, for Fig. 1B, 1D and 1E, this is not he case.
Specific points: 1. For COVID-19, both the pandemic (especially new mutants) and the research are changing very fast. Please update the related information in the revision. For example, the mutant that cause the surge of COVID-19 cases in India need to be discussed. 2. Except RBD, neutralization antibodies targeting NTD and S2 have also been identified. These should also be introduced. 3. Fig. 1B, D and E, concentration information are missing. 4. Line 52 "perhaps all coronaviruese...", how can the author claim "all"? 5. Line 149, what are the "RBM-mAbs" refer to? 6. Line 189, the injection volume needs to be calculated based on the weight of the hamster. 7. Is there a repetition of Fig. 2A to C? Please clarify. 8. It's not clear how the experiment was performed for Fig. 4D, please clarify. 9. Format (1) Page 5, line 138, pseudovirus particles (pp), the abbreviation has already given on Page 4, line 101, it does not need to explain again. Li and colleagues present a paper describing their efforts to isolate and characterize antibodies using a phage display library created from a SARS-CoV-2 (SARS2) convelescent patient. This effort led to the discovery of an antibody designated as FD20. Using functional and structural analyses they show that the antibody binds to the receptor binding domain (RBD) in a region that is outside of the ACE2 binding site (termed receptor binding motif or RBM in the manuscript). This antibody also has some cross-reactivity with SARS-CoV-1 (SARS1) and the binding location does not yet appear to be a mutational hot-spot within the RBD domain. The authors make 8 major claims in their report that at times are over-reaching in their conclusion or significance that would need to be addressed or modified.
A. The manuscript makes the following claims: 1. FD20 targets an uncharacterized epitope in RBD • mAb competition assays should be performed using antibodies with known epitopes to determine if the epitope is unique. 2. FD20 has ultra-high affinity (22pM) Comments: • The best practice for defining antibody affinity is to use titrate Fab or scFv (i.e., single binding arm) with a surface bound antigen. This minimizes avidity affects and allows for 1:1 fitting models to be applied to the acquire model. However, the claim of ultra-high affinity for FD20 is made using the full IgG molecule which is subject to avidity, which is not appropriate. Instead the appropriate affinity mentioned for the abstract and major claims should be 2.1 nM ( Figure 1B). • Even if IgG is used for affinity, while the affinity of 22pM is high ( Figure 1D), it is not unusual to have IgG affinities in the pM or sub-pM range. • The IgG affinity measurements ( Figure 1D/E) appear to have been calculated using a 1:1 fitting model. Since it is a bivalent interaction, the bivalent model should instead be applied. This result should have a k1on, k2on, k1off and k2off rates that is a measure of the avidity. • The methods do not indicate whether a global or individual curve fitting was performed for Figure 1B/D/E. This can impact the interpretation of the results. • In Figure 1B/D/E only the fitted curves are provided. The raw data curves should be shown in the same graphs. 3. FD20 is a potent neutralizer of B.1.1.7 and B.1.351 • The neutralization potency of the wt, B.1.1.7 and B.1.351 was stable but poor (i.e., 1.6 to 3.2 ug/mL) relative to other antibodies which are at least 100-1000-fold lower. Therefore, it is not appropriate to refer to FD20 as potent against any SARS-CoV-2 virus or variant. • Figure 2D shows various antibody neutralization IC50 and fold-change relative to wt neutralization for that antibody. The table is colored in heat-map fashion relative to the fold change for the antibody on a scale that starts from 0 to >1500. This results in a pink color for those that are 1-fold change or less and darker red color for those higher than 1-fold. This color scheme is misleading for several reasons. First, pink coloration for no change or increased potency could incorrectly suggest to the reader that potency is in fact worse. Second and more importantly, it does not account for variable initial potencies of the comparator antibodies. Therefore, antibodies that started with lower IC50 values than FD20 appear to be worse than FD20 when their IC50 is equivalent to it. For example, 10933 IC50 for B.1.351 is ~10-fold higher than wt but it's IC50 is lower than FD20. The color coding misleads the reader into thinking it is worse than FD20. • A minor comment on the table is that darker red colors make it difficult to read the black text. I would suggest using a white font for the darker colored boxes. 4. FD20 is modestly cross-reactive with SARS1 • This claim is supported by the results 5. FD20 reduces viral replication in hamsters • While the lung titer PCR is statistically significantly lower, it is less than 10-fold lower and there is no difference in Lung TCID50 and Nasal titer PCR or TCID50 (Figure 3). Stating the there viral replication is lower is over-reaching when considering the totality of the data. 6. FD20 binds to a site that that is ideal for low escape emergence • While using previously published data supports this idea, the authors should validate the critical contacts in the epitope using mutagenesis and assays for binding and neutralization • There are millions of sequences in the GSAID databased available for comparison. Therefore, only looking at 2-50 sequences ( Figure 4E and text) is a vast under sampling. The authors should take advantage of websites like cov.lanl.gov or others that can rapidly look at the residue in question and determine the true level of conservation. • Furthermore, the propensity for susceptibility to escape mutation was not formally tested. 7. FD20 neutralizes by destroying the spike • Prolonged incubations and fitting of models were used as evidence that FD20 destroys S. However, modeling is not proof. To more definitively show this they should perform EM analysis with immediately made grids and progressively increasing time 8. The "results reveal a conserved vulnerability site in SARS-CoV-2 Spike and perhaps all coronaviruses for the development of potential antiviral drugs." • The statement is overreaching and should be toned down. • See claim 6 above discussing conservation in SARS2. There should be more comparison for SARS1 and other coronaviruses B. In the virologic entry assays and order of addition experiment presented in Figures 5&6, significance claims are made for changes in FD20 responses that are statistically significant but likely not virologically significant. In entry assays, virologically significant changes are at least 5-fold but ideally 10-fold. Therefore, claims about order of addition, etc need to be tempered.
Minor: • Line 71: The distinction between the receptor binding domain and the receptor binding motif (RBM) may be confusing for some readers. It may be better to simply refer to the motif as the ACE2 binding site instead.
• Lines starting at 68 and remainder of paragraph 2 of the introduction: The manuscript uses a very simplified structural classification of RBD binding antibodies. Their classification scheme breaks up binding into those within the ACE2 binding site and those that bind outside the ACE2 binding site. However, this classification schema does not account for the known ability of the SARS2 RBD to be in two structural positions, the down (a.k.a. closed) or up (a.k.a. open) positions1,2. This is an important distinction because ACE2 can only engage with RBD is in the up position and some epitopes are partially or fully obscured with RBS is in the down position1,2. Thus, the field has generally chosen to use the Barnes classification3 that accounts for RBD position and antibody binding to the ACE2 site. This section and the manuscript would be improved if the Barnes classification was used. • Line 78: This reviewer needed to look up the definition of what the "core regions of the RBD" is. I would suspect others are not aware. Please define for the reader what this is. • Line 79: There are many antibodies that bind outside the ACE2 binding domain (Barnes Class III and IV) that are not crossreactive, so it is probably too strong to say "non-RBM mAbs are usually cross-reactive". I would suggest changing usually to often. • Line 124: Recommend avoiding terms like remarkable increase in activity rather say with that increase was. • Line 124-126: The improved potency from 119.4 to 11.9 nM is 10-fold better but this is not surprising given the increased avidity expected in a full IgG vs an scFv. Thus, it is not too remarkable a finding. • Line 139: did you mean to say 1.9-fold increase instead of 1-fold increase. • Lines 321-325. Why is the IC50 in the cell fusion assay so much different than the neut assays? Please define what you mean by "effective"? • Lines 340-344: Please provide or reference the data supporting the claim that RBM-mAbs have tight binding and slow dissociation. • Line 391: how is synergy determined • Lines 398-411. o It appears that S-2P protein was used. This should be explicitly used instead of S whenever appropriate.
• Throughout the manuscript there are word choices and sentence structure formats that cause the reader to have to infer or guess what is meant. Also, there are uses of words that are not usual, like iconic, that are not typical for scientific writing and may mislead the reader to either think the writers are making claims that I do not think they mean to. Another example on Line 256 uses the phrase "could inhibit" sounds like a hypothesis, but I think it is a statement. Therefore, the manuscript would benefit from being reviewed and edited by a professional English language scientific editor or someone similar who can provide the words and sentences that convey the authors meaning with more clarity.
The manuscript by T. Li et. al. reports isolation and characterization of a non-RBM monoclonal antibody (FD20) from COVID-19 convalescent patients with good neutralization activity against SARS-CoV-2. Although many neutralized Abs for SAR-CoV-2 have been reported before, the importance of FD20 is that it targets to a conserved epitope and neutralizes SARS-CoV-2 including the current concerning lineages B.1.1.7 and B.1.351. The authors have done experiments from different angels to prove the activities and potential mechanisms. I only have some minor concerns: We thank the reviewer for the positive comments and specific suggestions to improve our manuscript.
1. Fig. 1A： For the screening, the authors mentioned that B cells from three convalescent patients were pooled. Are there any criteria for choosing the convalescent patients? At the end of the screening, how many neutralized antibodies were obtained besides scFD20?
We performed a quick ELISA assay using RBD and chose three patients based on the ELISA results.
We obtained a total of 52 RBD-binding antibodies (as judged by their ability to shift gelfiltration peak of RBD), seven of which (including scFD20) showed >50% neutralization at 1 M concentration. The information has been included in the revised manuscript (Appendix Fig.  S1) and the sequences of the seven scFv antibodies have been included in Appendix Table S1. For your information, Appendix Fig. S1 is attached below. Figure S1. Identification of neutralizing antibodies. (A) Fifty-three scFv antibodies that bind to RBD on gel filtration were subjected to neutralization assays using SARS-CoV-2 pp and scFv at indicated concentrations.

Appendix
2. Line 184 We next investigated the in vivo potential of FD20 by intraperitoneal injection 6 h before intranasal inoculation of hamsters by live viruses (Figure 3): Is this sentence meaning that FD20 was injected first and then virus infection after 6h? It looks strange since normally for antibody treatment, the animals should be infected with virus first and then inject antibody for treatment.
The reviewer is correct that the hamsters were treated with the FD20 6 hours prior to intranasal challenge with SARS-CoV-2. Virus-neutralizing antibodies can be used prophylactically to prevent infection in high-risk cases, such as vulnerable individuals with underlying medical conditions which may not be vaccinated, health care providers, and individuals with exposure to confirmed cases of COVID-19.
We decided to use this experimental design as a proof-of-principle to determine the maximal effect of FD20. Because of the incomplete protection of FD20 and the limited available BLS3 slots during the ongoing SARS-CoV-2 crisis, we were unable to secure timeslots to test the efficacy of treatment at time points after initiation of infection to determine the therapeutic window.
3. Fig. 3B and 3E：The virus titers in either the lung or the nasal tissues determined by PCR and TCID50 were not consistent. For example, there are two lung tissues showing 0 TCID50 (Fig.  3C) and the PCR results ( Fig. 3B) are quite high. What could be the reasons behind? It is quite hard to understand why the titers were varied so much between the PCR method and the TCID50 method.
In general, some variability in viral titers/pathology etc. is to be expected, i.e., as no fully inbred strain of hamsters is available. Detection of SARS-CoV-2 is most sensitive and reproducible when using qRT-PCR, which is the data we base our conclusions on. However, in the context of potential for transmission, we also performed virus titrations on homogenates of lung and nasal turbinates, to detect the presence of infectious virus.
Consistently, the animals with the lowest RNA levels were also the animals in which infectious virus was not detected.
Unfortunately, the TCID50 method is less sensitive and due to regular toxicity of these homogenates in cell culture increases the limit of detection up to 1000 TCID50/mL. Therefore, while we could not detect infectious virus, in theory, there could still be up to 1000 TCID50 present in tissues. In addition to the issue described above, viral titers in nasal turbinates are even more variable due to the fact that we normalize all titers to grams of tissue. For lung tissue, we can typically take samples ~100 mg, which then only requires us to multiply by 10. However, in the case of nasal turbinates, 10 mg or less of tissue is typically harvested and thus increasing variability when normalizing to g tissue.

Referee #2
COVID-19, the pandemic caused by SARS-CoV-2, is still threatening thousands of millions of people around the world. In the absence of specific and highly effective medicine, the treatment of COVID-19 patient is very challenging, and neutralizing antibodies have great potential for treatment. There are many neutralization antibodies have been reported, most of them are targeting RBD, especially RBM, some are targeting NTD and S2. However, antibodies that directly against the RBM region may induce escape mutations. Therefore, more neutralization antibodies are still needed, especially antibodies that targeting conserved and functionally important regions on S protein, theoretically, which can reduce the occurrence of mutations and may exert a broad-spectrum antiviral effect against coronaviruses.
In this article, neutralizing antibody FD20 was isolated from COVID-19 convalescent patients through a phage display strategy. FD20 targets a conservative position and have a broadspectrum antiviral effect. For characterization and functional study, the authors demonstrated FD20's high affinity to ACE2, the effectiveness on cell-based neutralization assays. In order to understand the mode-of-action, the authors resolved the co-crystal structure of FD20-S protein.
Overall, the aim and main findings of this study are important, the design and logic are clear, the conclusions could be supported by the results. The manuscript is well-written. Thus, this reviewer endorses the publication of this work after revision.
We thank the reviewer for the complimentary remarks and specific comments below to improve our work.
Major points： 1. In most cases, the destination of a neutralization is for therapeutics. Thus, the in vivo (animal model) validation of its efficacy for protection or treatment is the key. Unfortunately, for animal model experiments, only marginal efficacy was observed. The authors need to improve the efficacy through practical ways, at least, the authors need to thoroughly discuss this.
We agree with the reviewer on this matter. We have tried to increase the binding affinity of FD20 by structure-based mutagenesis, an approach we have successfully used in our previous work with an RBD-targeting nanobody (Nat Commun 2021 12:4635). To this end, we designed 6 mutants (Fig R1 below) and compared their neutralizing activity with FD20. Unfortunately, the effort did not yield mutants with improved neutralizing activity (Fig R1). In the future, in vitro maturation techniques such as soft-randomization could be employed to obtain tighter mutants. Alternatively, FD20 may be fused to high-affinity mAbs or nanobodies to seek higher potency. A paragraph has been added to the Discussion as follows: Since the outbreak begins, thousands of neutralizing antibodies against SARS-CoV-2 have been reported (http://opig.stats.ox.ac.uk/webapps/coronavirus) and hundreds have their epitopes structurally characterized, most of which targets RBM and displays IC50 values 1-2 orders of magnitude 16,17,60 lower than FD20. For functional reasons, RBM is more exposed than other regions. Thus, the higher abundance of mAbs targeting the RBM is perhaps not unexpected. It is also possible that the non-RBM-targeting mAbs are generally less potent than RBM-targeting mAbs, and are thus less prioritized for study and less reported. Notably, the IC50 of FD20 (1. The in vivo protection of FD20 was also modest. This could be due to the relatively low neutralization activity in vitro. Alternatively, it may be related to half-life which is not tested in this study. Given that hamsters are becoming a common animal model for SARS-CoV-2 infection, and that little in vivo stability information is available for human mAb in hamsters, this issue warrants future investigation.
2. The in vitro affinity of FD20 to ACE2 is very high, while the in vivo efficacy is very low. A reasonable explanation is necessary? Is it possible that the in vivo stability of FD20 is low? Or FD20 was eliminated rapidly through some yet-to-be-discovered mechanism? The reviewer suggests the authors to test the distribution of FD20 in animal model at different time points.
As the reviewer #3 pointed out, the previously stated high affinity (22 pM) between the IgG FD20 and RBD is probably due to avidity effects. Instead, the affinity between the scFv version and RBD may be more appropriate, which is not very high (5.6 nM in the revised manuscript). Therefore, the low in vivo efficacy may be related to the modest affinity.
We considered the reviewer's comments about measuring the in vivo stability. However, our institute (Shanghai Institute of Biochemistry and Cell Biology or Institut Pasteur of Shanghai) does not have animal housing and protocols for Syrian hamsters. Therefore, regretfully, we did not do the experiments. Instead, we have added a paragraph to discuss this, as shown in the responses to the previous point.
3. For kinetic measurement, to assure the reliability of the determined Kd, the range of the serial dilution should cover the Kd. However, for Fig. 1B, 1D and 1E, this is not he case.
We thank the reviewer on this point. We have repeated the binding kinetics using lower concentrations until the BLI signal was too low to be reliable. We should note that the binding kinetics are now slightly different than previously reported. For your information, the new results are attached below (Fig 1B/C). We are trying to follow the development of Variants of Concern and we generated S derived from most of them. We have performed neutralization assays using SARS-CoV-2 Delta pp, as well as the Gamma pp. The results showed that FD20 can neutralize both variants with the same potency against the wild-type SARS-CoV-2 (1.0 fold for Gamma and 1.1 fold for Delta).
The results have been updated in Fig. 2D. 2. Except RBD, neutralization antibodies targeting NTD and S2 have also been identified. These should also be introduced.

Done.
3. Fig. 1B, D and E, concentration information are missing.
The following note has been added to the caption: Biolayer interferometry (BLI) assay was performed with RBD immobilized and scFD20 as analyte at indicated concentrations (nM). 4. Line 52 "perhaps all coronaviruese...", how can the author claim "all"? This overstatement has been removed in the revised manuscript.

Line 149, what are the "RBM-mAbs" refer to?
This inaccurate expression has been changed to "RBM-targeting" mAbs throughout the manuscript.
6. Line 189, the injection volume needs to be calculated based on the weight of the hamster.
In rodents, given the number of animals, typically the same amount of mAb is given assuming similar weights. We have revised the text to include the following: …as well as in viral RNA in the lungs of hamsters treated with FD20 (5 mg doses; 62-75mg/kg).
7. Is there a repetition of Fig. 2A to C? Please clarify.
Sorry for the confusion. There is no repetition. (D) Modest perturbation of scFD20 for RBD-ACE2 interaction. A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20containing buffer with (red) or without (blue) 25 nM of ACE2 for BLI signal recording. As a control, the ACE2-RBD interaction was monitored in the absence of scFD20 (black). 9. Format (1) Page 5, line 138, pseudovirus particles (pp), the abbreviation has already given on Page 4, line 101, it does not need to explain again.
We thank the reviewer for the careful reading. Done.

Referee #3
Li and colleagues present a paper describing their efforts to isolate and characterize antibodies using a phage display library created from a SARS-CoV-2 (SARS2) convelescent patient. This effort led to the discovery of an antibody designated as FD20. Using functional and structural analyses they show that the antibody binds to the receptor binding domain (RBD) in a region that is outside of the ACE2 binding site (termed receptor binding motif or RBM in the manuscript). This antibody also has some cross-reactivity with SARS-CoV-1 (SARS1) and the binding location does not yet appear to be a mutational hot-spot within the RBD domain. The authors make 8 major claims in their report that at times are over-reaching in their conclusion or significance that would need to be addressed or modified.
We thank the reviewer for the careful evaluation of our work, and constructive comments and specific suggestions below to avoid overstatements.
A. The manuscript makes the following claims: 1. FD20 targets an uncharacterized epitope in RBD • mAb competition assays should be performed using antibodies with known epitopes to determine if the epitope is unique.
When we first submitted our manuscript to other journals in September last year, we did competitive binding with the then available mAbs (REGN10933, REGN10987, CV30, CB6, CR3022) and we found that FD20 bound to RBD in the presence of other mAbs. Because the number of mAbs in the literature has grown very rapidly since, producing all mAbs with known epitopes (over 200 now) for competitive binding assays has become impractical. We have therefore removed this claim. The revised manuscript now focuses on the conservation of the epitope.
2. FD20 has ultra-high affinity (22pM) Comments: • The best practice for defining antibody affinity is to use titrate Fab or scFv (i.e., single binding arm) with a surface bound antigen. This minimizes avidity affects and allows for 1:1 fitting models to be applied to the acquire model. However, the claim of ultra-high affinity for FD20 is made using the full IgG molecule which is subject to avidity, which is not appropriate. Instead the appropriate affinity mentioned for the abstract and major claims should be 2.1 nM ( Figure  1B).
We now use the KD for the scFv only in the revised manuscript. Note that we have repeated the KD measurement using lower concentration as suggested by Reviewer #2, and the KD is now reported as 5.6 nM.
• Even if IgG is used for affinity, while the affinity of 22pM is high ( Figure 1D), it is not unusual to have IgG affinities in the pM or sub-pM range.
We have removed the results of the apparent binding kinetics information for IgG-RBD.
• The IgG affinity measurements ( Figure 1D/E) appear to have been calculated using a 1:1 fitting model. Since it is a bivalent interaction, the bivalent model should instead be applied. This result should have a k1on, k2on, k1off and k2off rates that is a measure of the avidity.
We have repeated the experiment using low concentrations of analyte as Reviewer #2 suggested and processed the data with both 1:1 and 1:2 fitting. The results (Fig R2 below) showed very low k2on. In addition, the 1:1 fitting matches the raw data better with a higher R 2 than the 1:2 fitting. The results may suggest low coating density and the minor existence of bridged complexes, although we do not have further evidence for this.
As the reviewer points out, the use of KD for scFv-RBD is more appropriate. Therefore, we have removed the results for the binding between IgG and RBD in the revised manuscript. We thank the reviewer for this comment. Global curve fitting was used for all binding kinetics. The information has been added in the Methods.
• In Figure 1B/D/E only the fitted curves are provided. The raw data curves should be shown in the same graphs.
Both the raw data and the fitted curve are now included in Fig 1B/C. 3. FD20 is a potent neutralizer of B.1.1.7 and B.1.351 • The neutralization potency of the wt, B.1.1.7 and B.1.351 was stable but poor (i.e., 1.6 to 3.2 ug/mL) relative to other antibodies which are at least 100-1000-fold lower. Therefore, it is not appropriate to refer to FD20 as potent against any SARS-CoV-2 virus or variant.
We have removed the claim that FD20 is a potent neutralizer and we have discussed ways to improve its potency in the Discussion.
• Figure 2D shows various antibody neutralization IC50 and fold-change relative to wt neutralization for that antibody. The table is colored in heat-map fashion relative to the fold change for the antibody on a scale that starts from 0 to >1500. This results in a pink color for those that are 1-fold change or less and darker red color for those higher than 1-fold. This color scheme is misleading for several reasons. First, pink coloration for no change or increased potency could incorrectly suggest to the reader that potency is in fact worse. Second and more importantly, it does not account for variable initial potencies of the comparator antibodies. Therefore, antibodies that started with lower IC50 values than FD20 appear to be worse than FD20 when their IC50 is equivalent to it. For example, 10933 IC50 for B.1.351 is ~10-fold higher than wt but it's IC50 is lower than FD20. The color coding misleads the reader into thinking it is worse than FD20.
We have modified the color coding to avoid misleading.
We now use cold colors for increased potency.
For the second issue, we now provide two heat maps: one for the fold change and one for the IC50 values to let readers judge with her/his criteria.
• A minor comment on the table is that darker red colors make it difficult to read the black text. I would suggest using a white font for the darker colored boxes.
We have removed the dark red color and the contrast is now more obvious. We thank the reviewer for the useful suggestion.

FD20 is modestly cross-reactive with SARS1
• This claim is supported by the results OK.
5. FD20 reduces viral replication in hamsters • While the lung titer PCR is statistically significantly lower, it is less than 10-fold lower and there is no difference in Lung TCID50 and Nasal titer PCR or TCID50 (Figure 3). Stating the there viral replication is lower is over-reaching when considering the totality of the data.
We agree with the reviewer that only a modest protective effect in the lung is observed in FD20treated animals. However, the significant reduction in viral RNA is supported by the significant reduction in viral antigen in the lungs. Although there was no significant difference in infectious titers, this is likely due to the large variation in viral titers, as 4 out of 5 animals had lower titers compared to PBS controls.
We have revised the text in the manuscript as follows: …as well as a small but significant reduction in viral RNA in the lungs of hamsters treated with FD20 (5 mg doses) compared to those that were untreated (p = 0.005) ( Figure 3B). 6. FD20 binds to a site that that is ideal for low escape emergence • While using previously published data supports this idea, the authors should validate the critical contacts in the epitope using mutagenesis and assays for binding and neutralization We have made 15 mutations for the 12 epitope residues and compared the binding kinetics and neutralizing potencies of the mutants with that for the wild-type ( Table 2). The results showed that 1) three mutants lost infectivity; 2) the binding affinity was variably affected by the mutations but the neutralization activity was not much affected; the two most dramatic mutants D426G and K462A had a ~2-fold IC50 compared to that of the wild-type.
For your information, the results are attached below: To further test whether the naturally occurring mutations affect FD20's binding and neutralizing activity, we have designed a panel of mutants as listed in Table 2 using the following criteria. For each epitope residue, the most frequent mutation was chosen unless the substitution was conservative, in which case amino-acid with the most dramatic changes was selected. For example, Arg466 was mutated to isoleucine instead of lysine. In addition, the charge of Lys462 and Glu465 were either eliminated by alanine mutation (not naturally occurring) or reversed by the mutations K462E and E465K (found in the database with low frequencies) (Fig EV1B), respectively; such mutations were expected to weaken the FD20-RBD binding because both residues provided intermolecular salt bridges (Fig 4B). Finally, the epitope-proximal residue Asp467 was mutated to tyrosine to test its structural/functional constraint. The mutations were introduced to RBD for binding assay separately with ACE2 ( Fig   EV2A) and FD20 (Fig. EV2B), and to S protein in SARS-CoV-2 pp for infectivity (Fig EV2C) and neutralization assay (Fig EV2D). In addition, the expression level and processing of S (whether S is cleaved to produce S1 and S2) were assessed by Western blotting as quality control for trafficking and maturation (Fig EV2E, EV2F).
Among the 15 tested RBD mutants, W353R, R355T, and D467Y showed no expression in insect cells ( Table 2). Although the corresponding full-length S mutants were detected in mammal cells, they were not processed effectively as evidenced by the lack of the S1 subunit on SDS-PAGE (Fig EV2E, EV2F). These results jointly suggest the mutants were misfolded.
As expected, mutations of Lys462 and Glu465 affected FD20-binding. However, the mutations either reduced infectivity drastically (E465K, 5% of the wild-type), or remained sensitive to FD20, displaying similar IC50 values (Fig. EV2D, Table 2). Similarly, mutation of Arg466, another residue that contributes a salt-bridge, also impaired binding but had little effect on the neutralization ( Table 2). Finally, the rest of the mutation did not weaken the binding activity, and the neutralizing activities of FD20 for these mutants also did not change noticeably ( Table   2). The two most escaping mutants were D428G and K462A, which showed a ~2-fold IC50 compared to the wild-type SARS-CoV-2 pp. Taken together, the analysis showed that the FD20 epitope is highly conserved, and the experimental results confirmed that some are indeed highly structurally (Trp353/Asp467/Arg355/Glu465) constrained as suggested by Starr et al. 38,54 ; in the case of mutants with similar infectivity to the wild-type, FD20 remained effective for neutralization, indicating FD20's potential to resist escape mutants.

Table 2. Summary of biophysical and biological characteristics of mutants at the FD20 epitope.
a Binding was performed with the wild-type or mutations on RBD, and infectivity and neutralization assays were performed with pseudoviruses harboring the wild-type or mutant S protein. b Frequency of the particular mutation followed by mutation frequency at this residue in brackets as of June 9, 2021 (www.gisaid.org; cov.lanl.gov). '0', no such mutation has been reported. d Binding properties were evaluated by the biolayer interferometry (BLI) signal and apparent binding affinity. c Not determined because either the construct had no expression in insect cells (for binding) or the mutant SARS-CoV-2 pp had low infectivity (for neutralizing assay). e Expression level of S was evaluated by Western blotting sing an anti-S1 antibody. f Proper processing and trafficking were evaluated by Western blot of S1 in the cell culture supernatant. Binding kinetics and expression level are rated from high to low following the sequence of "++", "++/-", "+", "+/-", and "-". The data for binding (BLI curve), expression (Western blot), infectivity, and neutralization are in Fig. EV2.
• There are millions of sequences in the GSAID databased available for comparison. Therefore, only looking at 2-50 sequences ( Figure 4E and text) is a vast under sampling. The authors should take advantage of websites like cov.lanl.gov or others that can rapidly look at the residue in question and determine the true level of conservation.
We thank the reviewer for pointing out this very useful resource to us. We have updated the   Fig. EV1. For your information, we have attached the relevant texts below: …Indeed, the mutation frequencies of the FD20 epitope residues from ~1.5 million deposited sequences 53 (cov.lanl.gov) were relatively low. Thus, there is only one mutation reported for Arg355 (R355T) (as of June 9 th , 2021), and the mutation frequencies (per million) of the rest residue were below 70, with the exception for Asn354 which was 391 (Fig EV1).

Figure EV1. Uneven distribution of naturally occurring mutants in RBD. (A)
The distribution of naturally occurring mutants in RBD viewed at different angles. Residues are color-coded according to their mutation frequency using the color scheme on the right. The FD20 epitopes are labeled as C spheres which are color-coded based on their interaction type with FD20 (by sidechain or by the main chain). (B) Details of the mutations of the FD20 epitope residues.
• Furthermore, the propensity for susceptibility to escape mutation was not formally tested.
We agree with the reviewer that this is an important experiment to do. The experiment would require the use of replication-competent viruses. However, we have been unable to locate resources for the experiment. We recognize the importance of this and we hope to test this in a follow-up study.
7. FD20 neutralizes by destroying the spike • Prolonged incubations and fitting of models were used as evidence that FD20 destroys S. However, modeling is not proof. To more definitively show this they should perform EM analysis with immediately made grids and progressively increasing time The incubation time was, at the time, chosen based on the conditions used in a previous publication for CR3022 (50 min and 3 h, Cell Host Microbe 2020 28:445). Besides, the fact that the control treatment did not cause S-destruction suggests that the 1-h incubation was unlikely an issue. Still, we think a time-course study would give more information. Therefore, we repeated the experiment and show that the S protein started to lose integrity within 3-15 minutes upon FD20 incubation (Fig. EV5). This information has been added to the revised manuscript, and as below: Regarding the model-fitting: We intended to convey that the model-fitting analysis was consistent with the experimental observation that FD20 destructs S. However, the previous writing might have led to the impression that we used model fitting as evidence that FD20 destructs S. We have now reorganized the paragraph to clarify this point (see below).

FD20 neutralizes SARS-CoV-2 by destructing Spike
Aligning the FD20-RBD structure to the full-length Spike structure showed that the FD20 epitope is inaccessible. As shown in Fig EV4A, all three FD20 binding sites are buried between the RBD and the N-terminal domain of S1 (NTD) of the adjacent monomers in the 'closed' state 56 when the three RBDs are at a 'down' conformation. When aligned to the 'open' state with one 'up'-RBD, the epitope is more exposed (Fig EV4B). Nevertheless, notable clashes are still observed between FD20 and NTD including the two glycans linking to Asn165 and Asn234 (Fig EV4C) 56 . This raises the possibility that FD20 executes its virucide effect by destructing S, as reported for CR3022 and EY6A 22,23 .
To test this hypothesis, we performed negative-stain electron microscopy of S-2P (an engineered S mutant containing two stabilizing proline mutations, see Methods) to investigate the impact of FD20 on the S-2P's integrity. The purified S-2P, but not FD20, showed particles with its typical 'chicken leg' shape (Fig 6C and 6D) which represents the side-views 56 . This feature was also clearly visible in the 2-D class averages. By sharp contrast, this feature was lost upon FD20 incubation in both the raw images and the 2-D class averages (Fig 6E). As a control, S-2P incubated with an unrelated IgG (5E1) 57 did not show loss of integrity (Fig 6F).
A time-course experiment showed that the S-2P protein started to lose integrity in the time window of 3-15 minutes upon FD20 incubation (Fig EV5). The results suggest that FD20 neutralizes SARS-CoV-2 mainly by destructing S. Because the FD20 epitope is structurally conserved in several other coronaviruses (Fig 4F), their corresponding region may be exploited for the development of neutralizing antibodies with a similar mechanism.
8. The "results reveal a conserved vulnerability site in SARS-CoV-2 Spike and perhaps all coronaviruses for the development of potential antiviral drugs." • The statement is overreaching and should be toned down.
We have changed the statement to "Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs".
• See claim 6 above discussing conservation in SARS2. There should be more comparison for SARS1 and other coronaviruses We have included the sequence alignment of RBD from SARS-CoV-2, SARS-CoV, MERS-CoV, hCoV-OC43, and hCoV-HKU1 (Appendix Fig S3C). These are coronaviruses known to infect humans with an RBD or RBD-like structure. In addition, RaTG13, a coronavirus highly similar to SARS-CoV-2, is included. The FD20 epitope is conserved between SARS-CoV-2, SARS-CoV, and RaTG13. For the rest of the three, the epitope residues are not conserved owning to the overall low sequence homology (16-21% identity). However, the epitope residues are organized similarly in 3-dimension when structurally aligned (Fig 4F).
The following paragraph has been added to the revised manuscript: …Complete conservation of the epitope residue was observed when comparing to RaTG13 ref.49 , of which the RBD is 89.5% identical and 94.0% similar to SARS-CoV-2 RBD (Fig. 4E).
Among five other coronaviruses known to infect humans, three of them (hCoV-OC43, hCoV-HKU, and MERS) contain RBD-like structures 50-52 . Although the sequence homology at the FD20's epitope was only modest due to their overall low sequence similarity (Appendix Fig  S3C), the 3-dimensional organization of the epitope region is structurally conserved (Fig 4F).
B. In the virologic entry assays and order of addition experiment presented in Figures 5&6, significance claims are made for changes in FD20 responses that are statistically significant but likely not virologically significant. In entry assays, virologically significant changes are at least 5-fold but ideally 10-fold. Therefore, claims about order of addition, etc need to be tempered.
We have modified the text to reflect the small virological changes: …When the mAbs were added after the binding step, an inhibition of entry, although modest in virological context (25%), was detected for FD20… Minor: • Line 71: The distinction between the receptor binding domain and the receptor binding motif (RBM) may be confusing for some readers. It may be better to simply refer to the motif as the ACE2 binding site instead.
We thank the reviewer for the suggestion to make the text less confusing but it is more convenient to use RBM in several cases, for example, 'RBM-targeting antibodies' instead of 'ACE2-binding site-targeting antibodies'. Therefore we respectfully ask to keep the term "RBM".
• Lines starting at 68 and remainder of paragraph 2 of the introduction: The manuscript uses a very simplified structural classification of RBD binding antibodies. Their classification scheme breaks up binding into those within the ACE2 binding site and those that bind outside the ACE2 binding site. However, this classification schema does not account for the known ability of the SARS2 RBD to be in two structural positions, the down (a.k.a. closed) or up (a.k.a. open) positions1,2. This is an important distinction because ACE2 can only engage with RBD is in the up position and some epitopes are partially or fully obscured with RBS is in the down position1,2. Thus, the field has generally chosen to use the Barnes classification3 that accounts for RBD position and antibody binding to the ACE2 site. This section and the manuscript would be improved if the Barnes classification was used.
Thanks to the reviewer for the suggestion. We have revised the introduction part according to the Barnes classification: The following sentence has been added to the revised Introduction.
"The RBD structure features a core region consisting of 5 -strands with connecting loops and -helices, and the RBM which is mainly made of loops and lay on top of the core region 9 ." • Line 79: There are many antibodies that bind outside the ACE2 binding domain (Barnes Class III and IV) that are not cross-reactive, so it is probably too strong to say "non-RBM mAbs are usually cross-reactive". I would suggest changing usually to often.
Done with thanks.
• Line 124: Recommend avoiding terms like remarkable increase in activity rather say with that increase was.
We thank the reviewer for this and other advice for scientific writing.
• Line 124-126: The improved potency from 119.4 to 11.9 nM is 10-fold better but this is not surprising given the increased avidity expected in a full IgG vs an scFv. Thus, it is not too remarkable a finding.
We have rephrased this and other similar expressions in the manuscript.
• Line 139: did you mean to say 1.9-fold increase instead of 1-fold increase.
• Lines 321-325. Why is the IC50 in the cell fusion assay so much different than the neut assays? Please define what you mean by "effective"?
We have modified the sentence as the following: Even at a 10 nM concentration, the cell-cell fusion was effectively suppressed by FD20, displaying a ~60% inhibition.
• Lines 340-344: Please provide or reference the data supporting the claim that RBM-mAbs have tight binding and slow dissociation.
The previous statement on this was incorrect. The binding affinity for the control mAbs are also in the nanomolar range: REGN10933, 3.4 nM; REGN10987, 45.2 nM (Science 2020 369:1010); CB6, 2.5 nM (Nature 2020, 584:120); CV30, 3.6 nM (Nat Commun 2020 11:5413). We have modified the sentence as the following: "…the weak pre-incubation effect by the control mAbs was probably due to residual antibodies that remained bound with pp." • Line 391: how is synergy determined As shown in Fig. EV3F, a mild synergistic effect of CV30 and FD20 was noticed by comparing the curve and IC50 of CV30+Y112R (14.3 nM, red curve) and CV30+FD20 (8.0 nM, black curve). This is measured using the black curve that was slightly shifted to the left following increased neutralization of the cocktail which provided a decrease in IC50 (8.0 nM). This result indicates first that the FD20 and CV30 are compatible and do not exclude each other, and, second, that the mixture of FD20+CV30 can help CV30 arrive at a better neutralization level, even if moderate".
We have added the following sentence to the caption of Fig. EV3: "The slightly higher neutralizing activity of CV30+FD30 compared with CV30+Y112R indicates modest synergy of the two mAbs (Y112R is a null mutant of FD20)." • Lines 398-411. It appears that S-2P protein was used. This should be explicitly used instead of S whenever appropriate.
It was indeed S-2P. Changes are made to reflect this.
• Throughout the manuscript there are word choices and sentence structure formats that cause the reader to have to infer or guess what is meant. Also, there are uses of words that are not usual, like iconic, that are not typical for scientific writing and may mislead the reader to either think the writers are making claims that I do not think they mean to. Another example on Line Thank you for the submission of your manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Please address all referee #3 comments. 2) We note that you currently have together with you, a total of 4 co-corresponding authors. Is that correct? Do you confirm equal contribution of these 4 people, able to take full responsibility for the paper and its content? While there is no limit per se to the number of co-corresponding authors, 3 is rare, 4 even more so, and may not reflect as intended to the community. 3) In the main manuscript file, please do the following: -Make sure that all special characters display well.
-In M&M, a statistical paragraph should reflect all information that you have filled in the Authors Checklist, especially regarding randomization, blinding, replication.
-In M&M, please specify the biosafety level for the experiments with SARS-CoV-2 by adding and amending the following sentence: All experiments with SARS-CoV-2 were performed in a ... level laboratory and with approval from... -In M&M, please include statement that the informed consent was obtained from all human subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
-In addition to the accession number please provide URL all deposited datasets. Please be aware that all datasets should be made freely available upon acceptance, without restriction. Use the following format to report the accession number of your data: The datasets produced in this study are available in the following databases: Please check "Author Guidelines" for more information. https://www.embopress.org/page/journal/17574684/authorguide#availabilityofpublishedmaterial 4) Funding: Please make sure that information about all sources of funding are complete in both our submission system and in the manuscript. 5) The Paper Explained: Please provide "The Paper Explained" and add it to the main manuscript text. Please check "Author Guidelines" for more information. https://www.embopress.org/page/journal/17574684/authorguide#researcharticleguide 6) Synopsis: -Synopsis text: Please provide a short stand first (maximum of 300 characters, including space) as well as 2-5 one sentence bullet points that summarise the paper as a .doc file. Please write the bullet points to summarise the key NEW findings. They should be designed to be complementary to the abstract -i.e. not repeat the same text. We encourage inclusion of key acronyms and quantitative information (maximum of 30 words / bullet point). Please use the passive voice.
-Please check your synopsis text and image, revise them if necessary and submit their final versions with your revised manuscript. Please be aware that in the proof stage minor corrections only are allowed (e.g., typos). 7) For more information: There is space at the end of each article to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc... 8) Source data: Please upload one file per figure. For blots or microscopy, uncropped images should be submitted (using a zip archive if multiple images need to be supplied for one panel). Please check "Author Guidelines" for more information. https://www.embopress.org/page/journal/17574684/authorguide#sourcedata 9) Press release: Please inform us as soon as possible and latest at the time of submission of the revised manuscript if you plan a press release for your article so that our publisher could coordinate publication accordingly. 10) Please be aware that we use a unique publishing workflow for COVID-19 papers: a non-typeset PDF of the accepted manuscript is published as "Just Accepted" on our website. With respect to a possible press release, we have the option to not post the "Just Accepted" version if you prefer to wait with the press release for the typeset version. Please let us know whether you agree to publication of a "Just accepted" version or you prefer to wait for the typeset version. 11) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF. The manuscript is highly improved and the vast majority of this reviewers concerns have been addressed. However, unfortunately the antibody's potency and breadth are lacking and the findings within the report do not represent a high level of novelty or impact.
There are still some issues if overreach but they have mostly been addressed. For example in the abstract, the claims for showing little to no escaping effect implies that escape experiments were performed. Instead it was an analysis of virus variants.
Other comments: 1) Figure 1C. The curve for the SARS1 protein kinetics show an upward shift near the transition point suggesting a complex binding interaction. This could indicate an issue with the loading and could lead to an incorrect value for the KD.
2) When discussing Figure 4E and the conservation of epitope residues it is not clearly written and can be confusing about whether it is the full RBD or RBD that is being referred to.
3) The supporting data for the new Table 2 should be provided

Referee #2 (Remarks for Author)
Is suitable for publication.
We thank the reviewer the supportive comments.
Referee #3 (Comments on Novelty/Model System for Author): The manuscript is highly improved and the vast majority of this reviewers concerns have been addressed. However, unfortunately the antibody's potency and breadth are lacking and the findings within the report do not represent a high level of novelty or impact.
We thank the reviewer for the constructive comments. We hope to improve the antibody's efficacy in follow-up studies.
There are still some issues if overreach but they have mostly been addressed. For example in the abstract, the claims for showing little to no escaping effect implies that escape experiments were performed. Instead it was an analysis of virus variants.
We have checked the manuscript thoroughly and re-phrased relevant texts to avoid misunderstanding as pointed by the reviewer.
In the Abstract, the sentence has been revised as: "Mutation of the residues of the conserved epitope variably affects FD20-binding activity but confers little or no resistance to neutralization." In the Results, the phrase 'the two most escaping mutants were D428G and K462A" were changed to 'the two most FD20-resisting mutants were D428G and K462A".
Other comments: 14th Oct 2021 2nd Authors' Response to Reviewers 1) Figure 1C. The curve for the SARS1 protein kinetics show an upward shift near the transition point suggesting a complex binding interaction. This could indicate an issue with the loading and could lead to an incorrect value for the KD.
We repeated the experiment by changing RBD loading, the buffer compositions, and by swapping the immobilized and the analyte. However, the upward shift persists. We suspect that the commercial SARS-CoV RBD may have been partially denatured, causing the abnormal binding profile.
We have modified the relevant texts as the following: "…Interestingly, scFD20 also bound with RBD from the closely related SARS-CoV (Fig 1C)." And we added a note in the caption of Fig. 1C: "Note, although the fitting of the binding curves in D gave a K D of 4.8 nM, the accuracy of the kinetic parameters may be compromised owing to the mismatch between the raw data and the fitted curves at the initial dissociation phase. The reason for the abnormal BIL profile is unknown but it may be caused by possible denaturation of the commercial SARS-CoV RBD during storage. It was also noted that the BLI signal in D was ~5 times less than that with SARS-CoV-2 RBD under similar conditions (C). " For your information, we have attached the results of the abovementioned experiments as below (Fig. R1, for review purposes only). 2) When discussing Figure 4E and the conservation of epitope residues it is not clearly written and can be confusing about whether it is the full RBD or RBD that is being referred to.
We have added a note to clarify this point.
"of which the RBD is 89.5% identical and 94.0% similar to SARS-CoV-2 RBD (Fig.   4E) (note, residues Pro330 and Lys529 of SARS-CoV-2 S was used to calculate sequence identity/similarity)." 3) The supporting data for the new Table 2 should be provided Done 18th Oct 2021 2nd Revision -Editorial Decision We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.

Reporting Checklist For Life Sciences Articles (Rev. June 2017)
This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript.

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.
definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).

The data shown in figures should satisfy the following conditions:
Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
The sample size for hamster experiments was 5 which would allow proper statistical analysis. This information is included under "Statistical analyses" in the Materials and Methods. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
We use animal size of 5 based on our previous experience.

The information is included under "Statistical analyses" in the Materials and Methods
No data were excluded in the analyses.

The statement is included under "Statistical analyses" in the Materials and Methods
Hamsters were randomly divided to experimental and control groups. This information is included under "Statistical analyses" in the Materials and Methods.
The sample size for the experiments are too small for normality test. However, they are assumed to have normal distribution. The exception was the data for virus titer. Because viruses growth follows an expolential distribution, the data were mathematically transformed using Log-function to approximate normal distribution (J Clin Bioinforma 2012 2:5; Proc Natl Acad Sci USA 2004 101:8682).
The information are included under "Statistical analyses" in the Materials and Methods.
Animals were randomly divided into experimental and control groups. This information is included under "Statistical analyses" in the Materials and Methods.
For histopathology and immunohistochemistry, the following statemnt is the manuscript: "Sections were examined without knowledge of the identity of the hamsters." The statement is also included in the "Statistical analyses" section.
The pathological analysis was performed without knowing the identity of the experimental/control groups. Other analysis was carried out without blinding as the readout are considered to be not influenced by the prior knowledge. This information is included under "Statistical analyses" in the Materials and Methods.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.