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After revisions, all reviewers agreed to publish the manuscript. I also reviewed the manuscript and found no obvious risks to publication. Therefore, I also approved the publication of this manuscript.
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
The authors addressed my previous comments very well.
The authors added references and improved their figure quality.
The authors expanded their discussion on the results and cited relevant studies.
Overall, the authors made improvement on their manuscript to meet the standard of the journal. I recommend the article for acceptance.
The authors have answered my questions and adjusted the paper accordingly. The quality of the figures has also been improved. The writen English was alos improved. They also refined the format and citation. The paper is now ready for publishing.
The authors have addressed my concerns and made sufficient modifications to the paper. I am satisfied with the current experimental design.
1. I don’t agree with the authors' choice of Swiss-Model over AlphaFold. The authors' answer is unconvincing. However, I will let this pass. Next time, the authors should provide evidence that Swiss-Model is superior to AlphaFold in their study. For homology modeling, there are better options like Rosetta. If the authors have time, they can read the following articles about protein structure prediction (DOI: 10.1038/s41592-020-0848-2, 10.1126/science.abj8754, 10.1038/s41586-024-07487-w).
2. The explanation I was looking for is that a positive binding affinity means the binding is not spontaneous and requires extra energy from the environment, while a negative binding affinity means that the binding is spontaneous and releases energy into the environment. However, I am fine with the authors' answer.
The authors' response is convincing. They have provided sufficient evidence to support their conclusions. The experimental results are clearly presented, and the data analysis is thorough. Overall, the study's findings are valid and contribute valuable insights to the field.
The authors are requested to carefully revise the manuscript and answer the questions raised by the reviewers.
The authors improved their manuscript and addressed my previous questions. My only suggestion is that the authors should annotate or name the files better in the Raw_data.zip file.
Grammar error in line 320 "lower their toxicity and reduces the immune escape". I see the authors have already made some inquiry about language editing to the editor in the response letter.
The authors made proper revision and provided additional information to address my previous concerns. The authors also replied that they could not disclose the protein sequences used in the study due to a patent application.
I still have two minor suggestions.
1. The authors should cite proper references in addition to providing the website link for the computational tools.
2. I still find the resolutions a bit too low for some figures such as Fig 4C and Fig S2. Some of the labels are hard to read. Maybe the editor can decide whether the resolution satisfies the standard for the journal.
Since the title of the manuscript focuses on sdAbs but the conclusion also points out the potential of fusion proteins, my minor suggestion is that the authors should expand their discussion a bit more (including any concerns) when comparing the bivalent or multivalent nanobodies and their fusion protein constructs with sdAbs. Is the claim in line 319 and 320 supported by any studies?
The author made sufficient revisions and improved the quality of the manuscripts. Overall, the manuscript is suitable for publication.
The manuscript, "Screening and affinity optimization of single-domain antibody targeting the SARS-CoV-2 nucleocapsid protein," shows that anti-SARS-CoV-2 fusion proteins have the potential to be used in antigen detection kits for SARS-CoV-2. The idea is novel, but the paper has some problems that need to be addressed before it can be considered for acceptance.
1.The writing is inconsistent. Line 22, 37, 79 start with tabs, but line 48,57, and 71 didn't. What are the reasons behind this?
2.After the first two reviewers mentioned the resolution problem, the quality of the figures still does not meet the publisher's requirements. For example, it is impossible to tell the length of the hydrogen bonds from the Ligplus figures.
3.Line 220-227, the description of hydrogen bond interactions is too dry. Please provide a table to demonstrate the relationships among the ligand, receptor, binding affinity, and the number of non-covalent bonds.
Additionally, for lines 234-236, I believe the binding affinity is -9 rather than 9. 9 and -9 are different concepts. Please make sure the author understand the concept of binding affinity.
4.Regarding academic ethics, the author used Ligplus without citing DOI: 10.1021/ci200227u in the references section. This is considered plagiarism. It's not the reviewer's duty to check your references. Please keep that in mind. Same for other softwares or web servers. Please refine the citation.
1.For the method, why did the author use Swiss-Model rather than AlphaFold2 or AlphaFold3? During CASP14, AlphaFold2 outperformed other methods (DOI: 10.1038/s41586-021-03819-2). It is important to state the reasons for all your choices. Additionally, AlphaFold3 is more popular than Hawk Dock.
2.The sdAbs show the binding affinity in Table 3. To be consistent, the fusion proteins should also show their affinity values.
1.The result is convincing. However, the author needs to prove that the anti-SARS-CoV-2 fusion proteins only bind to SARS-CoV-2 before claiming that the anti-SARS-CoV-2 fusion proteins can be used as antigen detection kits for SARS-CoV-2.
The authors are requested to revise the manuscript as requested by the reviewers and to answer questions.
**PeerJ Staff Note:** Please ensure that all review and editorial comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
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**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors agree that they are relevant and useful.
(1) The present works lacks a proper background introduction to earlier studies on the same subject and the general field. For example, doi: 10.3389/fimmu.2021.719037 and doi: 10.1021/acs.analchem.1c00677 both developed sdAbs for the N protein. How does the present work compare to previous studies and contribute to the field? Relevant discussion is also lacking in the manuscript.
(2) The authors did not properly provide the raw data. The raw data provided in the supplemental materials is not well organized as the sequence information for the sdAbs or the fusion protein could not be found. There are additional files in the raw data that cannot be read or opened directly.
(3) The data deposition statement include protein PDBs not from this work. I believe the authors might have misinterpreted the purpose of this statement as they did not mention the sdAbs or the fusion protein from their present work here.
(4) The English language used in the paper could be improved. There are redundant texts (line 51, 53) and grammatical errors, e.g., line 74, 75. There are also a few confusing places in the discussion section where the text cannot be clearly understood, e.g., line 260.
(5) The authors might have provided inaccurate references. In line 57, the authors cited [4,5] to argue that N protein is a suitable target. However, SARS-CoV-2 nucleocapsid protein has never been mentioned in the papers [4,5].
(6) The authors provided molecular characterization of the sdAbs but did not provide sufficient interpretation of their molecular docking results, and the figures provided are too blurred to tell the interaction.
(7) In addition to what’s mentioned above, the authors did not provide any details about how the molecular docking was performed and how much one should believe such models without any structural validation. All the competitive binding hypotheses need to be validated by experiment.
(8) The authors mentioned statistical analysis in the Methods, but I could not find any data presented in the tables with statistical errors.
(9) In general, I found the interpretation of the results insufficient. The authors showed the sdAbs can bind to the N protein and tried to improve their affinities using fusion proteins. Despite the higher affinities the authors argued for the fusion proteins, the authors failed to address any concerns for the larger size and worse accessibility of the fusion proteins compared with sdAbs. Additionally, Figure 4A shows pN3-1-3 as two separate chains.
(10) In the discussion, the authors tried to “align” each of the sdAbs with different antibodies. I found this part to be quite confusing. The authors should discuss how their sdAbs compare with the sdAbs in previous studies.
(11) The final conclusion is not well supported as the current work did not show whether the sdAbs or the fusion proteins would work in real diagnostic and therapeutic applications where data such as detection limit is often required.
In this paper, the authors aimed to develop single domain antibodies (sdAbs) targeting SARS-CoV-2 nucleocapsid (N) protein. The authors identified a few candidate sdAbs as well as fusion proteins that can bind to the N protein in vitro. However, the conclusion of the paper that these sdAbs or fusion proteins are suitable for diagnostic applications is not well supported by the results presented. The current state of the manuscript does not meet the standards expected of the journal. The issues mentioned above should be carefully addressed before the paper can be further considered for acceptance.
The manuscript entitled "Screening and aûnity optimization of single domain antibody targeting the SARS-CoV-2 nucleocapsid protein" is novel and well-organized. It provides a new target for the early diagnosis of Covid-19. I only have several minor comments.
The experiment design is clear and with good logistic flow. The authors extensively introduce the basic knowledge of what COVID-19 and SARS-CoV-2 are, but the introduction of the limitation of the current diagnosis method is missing. The sensitivity and specificity tests are also missing.
The results are solid and well-presented, but the resolution of several figures is too low, such as Fig. 1 C, D, and Fig. 3 A. It's tough to read the text.
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