Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Variant-Specific Monoclonal Single-Domain Antibodies from a Synthetic Library

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study reports the rapid discovery of XBB.1.5- and JN.1-specific single-domain antibodies (sdAbs) from an alpaca-derived synthetic library, demonstrating high specificity, nanomolar to sub-nanomolar affinities, ACE2 inhibition, and practical applicability in vaccine strain identification assays. The manuscript  is important because it addresses a critical bottleneck in SARS-CoV-2 vaccine quality control by providing a fast, immunization-free strategy to generate strain-specific reagents aligned with the accelerated timelines of seasonal vaccine updates.

1. The Abstract should diferenciates  diagnostic/quality-control utility  from  therapeutic relevance , abecause  ACE2 inhibition may be misinterpreted as implying neutralization in vivo, which is not experimentally demonstrated.
2. In the introduction , I sugesst to more y state earlier in the introduction in a more explicitl way, that  the primary goal is an analytical assay development , not therapeutic antibody discovery
3. In the Materials and Methods the  criteriums for clone down-selection  (beyond “specificity, affinity, and uniqueness”) should be more quantitatively defined.
4. For HDX-MS, the rationale for the >10% differential uptake threshold  should be briefly justified or referenced.
5. The statistical analysis is adequate, but authors should report the replicate numbers for key functional assays (ELISA, ACE2 inhibition) .
6. A minor concern is that functional ACE2 inhibition is shown only in vitro , and its relevance to vaccine identification (as opposed to neutralization) should  be more clearly contextualized.

 

7. In the discussion the authors should moderate their statements suggesting broader implications for monoclonal antibody discovery, because the demonstrated strength of this work lies primarily in  analytical reagent development rather than therapeutic advancement .

 

8. In the conclusions as mentioned above the authors should explicitly acknowledge the analytical (non-therapeutic)  scope as a defining feature and advantage of the approach.

 

Author Response

Response to the reviewer’s comments:

Manuscript ID: antibodies-4108833: “Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Vari-ant-Specific Monoclonal Single Domain Antibodies from a Synthetic Library”

Reviewer 1

1. Summary:

Thank you very much for your helpful feedback. We appreciate your input and agree that the article should concentrate more on tool antibody discovery not for therapeutics. We have revised the article according to your suggestions.

 

2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Is the research design appropriate?	Yes
Are the methods adequately described?	Can be improved	Agree and add more detailed information for assay numbers
Are the results clearly presented?	Yes
Are the conclusions supported by the results?	Can be improved	Agree: add introduction and rationale for tool antibody discovery
Are all figures and tables clear and well-presented?	Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The Abstract should differentiates diagnostic/quality-control utility from therapeutic relevance , because ACE2 inhibition may be misinterpreted as implying neutralization in vivo, which is not experimentally demonstrated

Response 1: Thank you for your comment. We revised the abstract to highlight the vaccine identification assay and removed the ACE inhibition section. Line 31, 40-41 46-47  

Comments 2: In the introduction, I suggest to more y state earlier in the introduction in a more explicit way, that the primary goal is an analytical assay development , not therapeutic antibody discovery

Response 2: Thank you for your feedback. We have condensed the antibody discovery section to an introduction and shifted our focus to vaccine assay development (Lines 70-74).

Comments 3: In the Materials and Methods the criteriums for clone down-selection (beyond “specificity, affinity, and uniqueness”) should be more quantitatively defined.

Response 3: Thank you for bringing this to our attention. We acknowledge your comment and have incorporated screening criteria for down selections. Please note that further details cannot be disclosed due to confidentiality considerations. Line 120-125

Comments 4: For HDX-MS, the rationale for the >10% differential uptake threshold should be briefly justified or referenced.

Response 4: Thank you for your comment. We have added reference 27 in Line 216 to clarify the 10% threshold.

Comments 5: The statistical analysis is adequate, but authors should report the replicate numbers for key functional assays (ELISA, ACE2 inhibition) .

Response 5: Thank you for your feedback. We have added the replicate numbers for each experiment in Lines 155-157, 165-166, 181-182, 256-257, 267-270, 272-273, and 299-301.

Comments 6: A minor concern is that functional ACE2 inhibition is shown only in vitro, and its relevance to vaccine identification (as opposed to neutralization) should be more clearly contextualized.

Response 6: Thank you for your feedback. We agree and add a comment to Line 87-88: neutralizing activities are not required for its mAb character.

Comments 7: In the discussion the authors should moderate their statements suggesting broader implications for monoclonal antibody discovery, because the demonstrated strength of this work lies primarily in analytical reagent development rather than therapeutic advancement.

Response 7: Thank you for your comment. We have added an introduction to therapeutic and tool mAbs in the laboratory section and biologics production assays Lines 341-349.and change the structure to discuss the tool sdAb discovery for vaccine identification earlier. Lines 358-363

Comments 8: In the conclusions as mentioned above the authors should explicitly acknowldge the analytical (non-therapeutic) scope as a defining feature and advantage of the approach.

Response 8: Thank you for your feedback. We revised the conclusion to address the approach's analytical scope for vaccine and biologics. Lines 410-415.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, Tsuji I, et al report the use of alpaca-derived synthetic antibody libraries to rapidly identify XVV.1.5- and JN.2-specific single-domain antibodies (sdAbs) within 8-9 weeks and demonstrate their application in vaccine product identification assays. The study addresses an important need for rapid strain-specific reagent development and has clear potential utility for accelerating vaccine development workflows.

1. On line 120, it is unclear why the XBB.1.5 selection was performed using only the Wuhan RBD protein, whereas the JN.1 selection used Wuhan, BA.5, and XBB.1.5 RBD proteins. A clearer explanation of the rationale behind these different selection strategies would help readers better understand the basis for strain specificity.
2. Line 221: For clarity, the author may consider explicitly stating the total number of clones screened and the selection criteria applied at each stage of the 4-week discovery process. 

3. Given that both anti-XBB.1.5 and anti-JN.1 sdAbs have extended CDR3 regions, why is strong strain specificity and Fc-dependent affinity enhancement observed primarily for anti-JN.1 sdAbs?

4. Why does Fc fusion enhance affinity for anti-JN.1 sdAbs but not for anti-XBB1.5 sdAbs, and what does this suggest about epitope accessibility or binding geometry?

5. How generalizable is this alpaca sdAb-based approach for rapid, strain-specific assay development against future SARS-CoV-2 strains?  

Author Response

Response to the reviewer’s comments:

Manuscript ID: antibodies-4108833: “Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Vari-ant-Specific Monoclonal Single Domain Antibodies from a Synthetic Library”

Reviewer 2

• Summary:

Thank you very much for your insightful feedback. We sincerely appreciate your suggestions and constructive comments. Many of your points offer significant scientific perspectives and highlight important issues. However, since our primary focus is on antibodies as tools for vaccine development, we do not provide an in-depth analysis comparable to that of therapeutic antibodies. Nonetheless, I will share my responses and thoughts to address your queries.

 

2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes
Is the research design appropriate?	Can be improved	Add explanation how to select counter proteins. (previous prepare vaccine strain)
Are the methods adequately described?	Can be improved	Add more detailed information for assay numbers
Are the results clearly presented?	Yes
Are the conclusions supported by the results?	Can be improved	Explain more about the rationale for tool mAb discovery
Are all figures and tables clear and well-presented?	Can be improved	Add explanations in the legend part

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: On line 120, it is unclear why the XBB.1.5 selection was performed using only the Wuhan RBD protein, whereas the JN.1 selection used Wuhan, BA.5, and XBB.1.5 RBD proteins. A clearer explanation of the rationale behind these different selection strategies would help readers better understand the basis for strain specificity.

Response 1: Thank you for your comment. Counter-selectors refer to RBD proteins from previously prepared vaccine strains. For XBB.1.5 sdAb discovery, only Wuhan was used as a vaccine strain, while Wuhan, BA.5, and XBB1.5 were included for JN.1 sdAb discovery. We have now clarified this on Lines 118-119.

Comments 2: Line 221: For clarity, the author may consider explicitly stating the total number of clones screened and the selection criteria applied at each stage of the 4-week discovery process.

Response 2: Thank you for your feedback. We have added the screening criteria and selected numbers to Lines 120-125.

Comments 3: Given that both anti-XBB.1.5 and anti-JN.1 sdAbs have extended CDR3 regions, why is strong strain specificity and Fc-dependent affinity enhancement observed primarily for anti-JN.1 sdAbs.

Response 3: Thank you for bringing this to our attention. We agree with your comments; however, it is difficult to address your question in the manuscript. Unfortunately, we are unable to explain why the affinity differs so greatly. My speculation is that the alpaca sdAb framework might be better matched to the JN.1 RBD structure than to the XBB.1.5 structure. I added the sentences the importance to find the differences between two sdAb for future sdAb discovery Line 388-394

Comments 4: Why does Fc fusion enhance affinity for anti-JN.1 sdAbs but not for anti-XBB1.5 sdAbs, and what does this suggest about epitope accessibility or binding geometry?

Response 4: Thank you for your feedback. While we agree with your comments, it is challenging to fully address your question in the manuscript. Briefly, I believe the increased affinity of sdAb-Fc fusion protein results from the avidity effect therefore, I added the reference [28] and explained the change for JN.1 sdAb Line 280. But we cannot explain why the XBB.1.5 clone did not show this enhancement. My speculation is that the avidity effect requires a certain level of affinity; higher affinity (e.g., JN.1) allows enhancement, whereas lower affinity (e.g., XBB.1.5) may be insufficient.

Comments 5: How generalizable is this alpaca sdAb-based approach for rapid, strain-specific assay development against future SARS-CoV-2 strains?

Response 5: Thank you for your comment. We have established strategies for SARS-CoV-2 strain antibody discovery and have identified new sdAbs for 2024-2025 strains using these methods. We also recognize unmet needs for tool antibodies in biologics production, which we discuss in Lines 341-348. Our approach is applicable not only to vaccine assays but also to tool antibodies for biologics, in summary section in Lines 410-415.

 

Author Response File: Author Response.pdf