PPATHOGENS-D-25-02460

Directed evolution of a stem-helix–targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity

PLOS Pathogens

Dear Dr. Andrabi,

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Kind regards,

Mario Juan Borgnia, Ph.D.

Guest Editor

PLOS Pathogens

Ronald Swanstrom

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Panpan Zhou, Meng Yuan, Yuexiu Zhang, Oliver Limbo, Ge Song, Fangzhu Zhao, Hejun Liu, Wan-ting He, Tazio Capozzola, Sean Callaghan, Gabriel Avillion, Xuduo Li, Nathan Beutler, Peter Yong, Fabio Anzanello, Thomas F Rogers, Dennis R Burton, Joseph G Jardine, Ian A Wilson, and Raiees Andrabi. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors describe the isolation of CC65.1, a stem-helix targeting antibody, and its subsequent affinity maturation to generate variants (e.g., eCC65.1) capable of neutralizing MERS-CoV. The study is well-conceived, and the use of directed evolution to broaden the neutralizing breadth of antibodies targeting cryptic epitopes is of high interest to the field. The manuscript is well-written, and the structural analyses provided are robust. However, a few technical details regarding the spike constructs and mechanism of action need clarification to ensure a comprehensive understanding of the discovery.

Reviewer #2: In this article titled “Directed evolution of a stem-helix–targeting antibody enables MERS-CoV crossneutralization through enhanced binding affinity” the authors Zhou, Yuan, Zhang, Limbo et al. describe the in vitro affinity maturation of an antibody to gain neutralization activity against MERS-CoV. Starting from an antibody that bound to the MERS-CoV stem helix region but did not neutralize the virus, the authors performed directed evolution targeting the antibody CDRs coupled with high throughput screening with antigen to obtain an antibody that was able to neutralize MERS-CoV, while also retaining neutralizing activity against other betacoronaviruses. Crystal structures in complex with antigen were determined for both the starting and the engineered antibody enabling high resolution understanding of the binding mode of the antibody as well as the impact of the mutations in the evolved antibody. Overall this study reports important data and highlights how engineering existing antibodies can allow expansion of neutralization breadth – this is of value for developing broad reactivity to Coronaviruses and responding to future pandemics.

I am in support of publication of this manuscript in Plos Pathogens once the authors have had a chance to address the following:

1. The CC65 serum neutralizes MERS-Cov (Figure 1A), yet the antibodies isolated from the serum do not neutralize MERS-CoV, which likely means there is a yet undiscovered antibody (or antibodies) in the serum that neutralizes MERS-CoV. Could the authors comment on this in the discussion?

2. Lines 174-175: “To confirm these epitopes recognized by CC65.1 and verify whether these residues are crucial for CC65.1 neutralization,…” The crystal structure has already confirmed that these epitopes are recognized by the antibody. The mutagenesis and binding/neutralization assays provides functional context to the observed contacts. I recommend revising the sentence.

3. The affinity measurements reported are in the context of avidity because of the multivalent analytes and the authors appropriately designate these as apparent affinities. While not as rigorous as reporting intrinsic affinities using Fabs as analyte, the measurements reported seem sufficient for the goals of this study. Recommend adding a sentence clarifying the use of apparent affinity.

4. Figure 3 C and D: There is no description in the figure or in the figure legend for what the pink, yellow and orange dots are. Assume they are the antibodies shown in S6C. I recommend color coding the antibody names in Figure S6C, and using these colors in Figure 3 C and D (and in 3F), ie., use discrete colors for each variant.

5. BLI sensorgrams should be shown in a Supplemental figure for S6C.

Minor:

1. Lines 108-109: “Except stem helix region, sequence identify between SARS-CoV-2 and HCoV-HKU1 (embecovirus) spike is low.” Change “identify” to “identity”.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. Mechanism of Neutralization: The manuscript lacks a discussion on how affinity maturation enables neutralization of MERS-CoV when the parent antibody binds but does not neutralize. The current explanation treats affinity as a standalone metric. The authors should explicitly connect their hypothesis on accessibility (Lines 258-260) to the neutralization data. Does the high affinity of eCC65.1 allow it to "catch" the transiently exposed stem helix on MERS-CoV more effectively, thereby increasing occupancy and blocking the hairpin formation in the fusion machinery? This complexity of epitope accessibility is further highlighted by the SARS-CoV-1 data. Lines 121-122 claim "stronger binding" for SARS-CoV-1 in "both formats," yet CELISA data (Fig S1B) shows significantly weaker binding for SARS-CoV-1 compared to SARS-CoV-2, despite identical epitope sequences. Discussing this "occupancy model" is necessary to resolve the apparent paradox of why the parent antibody binds but fails to neutralize.

2. Clarification of Spike Constructs: The text fails to explicitly state whether WT, S-2P, or HexaPro constructs were used for the specific spike proteins (SARS-CoV-1/2 vs. MERS-CoV) in the binding assays (SPR/BLI). Please provide these details in the Methods section.

3. Terminology: The authors frequently use the term "rational engineering." However, the approach utilized is directed evolution, not rational design. In a typical rational design approach (e.g., structure-guided), one would target the direct interface residues to improve affinity. In contrast, the key impact here appears to come from paratope stabilization between CDRL1 and L2, potentially reducing the entropic cost of capturing a transiently breathing epitope. Notably, the interacting residues are effectively the same (with the exception of the S93A mutation), while the significant improvements arise from distal stabilization (e.g., the Lys-His interaction stabilizing the Tyr rotamer). I recommend reframing the text to highlight that directed evolution was capable of identifying this sophisticated, non-intuitive structural solution, emphasizing the power of the method used.

Reviewer #2: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Breadth of Screening (Line 103): The text states sera were screened against betacoronaviruses "including SARS-CoV-2 and MERS-CoV." This phrasing implies a broader panel (e.g., Lineage A). If only these two were tested, the text should be precise to avoid overstating the breadth.

Figure 1E: SARS-CoV-1 and SARS-CoV-2 are represented by a single curve. While it is implied that they share an identical stem helix sequence, this should be explicitly stated in the figure legend.

Reviewer #2: (No Response)

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Reviewer #1: Yes: Arvind Sharma

Reviewer #2: No

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Dear Dr Andrabi,

We are pleased to inform you that your manuscript 'Directed evolution of a stem-helix–targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity' has been provisionally accepted for publication in PLOS Pathogens.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Mario Juan Borgnia, Ph.D.

Guest Editor

PLOS Pathogens

Ronald Swanstrom

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: (No Response)

Reviewer #2: The authors have adequately responded to reviewer critiques. I recommend publication of this manuscript.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: arvind sharma

Reviewer #2: No