Antibodies Against SARS-CoV-2 Nucleocapsid Protein Possess Autoimmune Properties

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript and for a positive feedback. Please find the detailed responses below and the corresponding revisions/corrections highlighted in red in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Can be improved

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Can be improved

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

The study of Alexandra Rak et al addresses an important topic: the potential autoreactivity of antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Given the widespread exposure to SARS-CoV-2 and the growing interest in N-based vaccines, understanding potential autoimmune consequences is of high relevance. The study integrates Western blotting, MALDI-TOF mass spectrometry, and structural bioinformatic analyses to identify possible human autoantigens recognized by anti-N antibodies generated in different animal models.

Comments 1: Is it possible to have the densitometric analysis of the blots?

Response 1: We thank the reviewer for this suggestion. Indeed, densitometric analysis of the blots could provide some additional information on the content of each protein/band relative to the other proteins within the sample or between samples on the same blot. However, the most informative comparison would be when anti-N sera is compared with the naïve or biological control sera, but these bands come from different blots and direct comparison would not be correct enough. We want to emphasize that our study is more qualitative rather than quantitative. Nevertheless, we have included this point in the study limitations: “Furthermore, since this paper reports on the findings of preliminary experiments, we did not perform WB densitometry or investigate the ability of autoreactive anti-N antibodies of various origins to bind to the purified or recombinant human-derived proteins. Consequently, the results have not undergone validation.”

Comments 2: The inclusion of anti-influenza NP immune sera as a negative control is valuable. However, it would also be important to clarify why the authors chose NP specifically — e.g., because NP and N share similar structural features as internal viral proteins?

Response 2: Thank you for pointing this out. In according to the comment, we have added a description of the biological control choice in the 3.1 subsection: “This antigen has been chosen as sharing similar structural features with N protein as internal viral component. In addition, anti-NP antibodies are actively induced upon influenza infection and immunization with live attenuated or inactivated whole virion influenza vaccines [29,30], with no safety signals of developing autoimmune consequences after infection or vaccination.”.

Comments 3: The study (Front Immunol 2023; DOI: 10.3389/fimmu.2023.1129201) provides direct evidence that COVID-19–associated antiphospholipid antibodies activate platelets via ERK/p38 signaling, contributing to a hypercoagulable and proinflammatory state. Because the manuscript discusses autoimmune mechanisms, post-COVID complications, and the possible pathogenic effects of non-neutralizing antibodies, inclusion of this reference could help reinforce the clinical and mechanistic relevance of the authors’ observations.

Response 3: We thank the reviewer for this important note. As suggested, the reference has been mentioned in the Discussion section: “The similar ability of non-neutralizing COVID-19–associated antibodies to provoke the pathogenic effects like hypercoagulable and proinflammatory state has been described previously [60,61].”

Comments 4: The authors could consider the review Front Cell Dev Biol 2021; DOI: 10.3389/fcell.2020.618296 which provides an in-depth discussion of lipid rafts in coronavirus entry, immune modulation, and the membrane localization of viral proteins.
Given that the manuscript highlights the presence of nucleocapsid protein on the cell surface and its potential immunological implications, incorporating this reference could help strengthen the mechanistic framework regarding the role of membrane microdomains in SARS-CoV-2–related immune processes.

Response 6: Thanks a lot for raising an interesting question. We agree with the feasibility of the text deepening, and therefore have added the reference description in the Discussion section: “The similar ability of non-neutralizing COVID-19–associated antibodies to provoke the pathogenic effects like hypercoagulable and proinflammatory state has been described previously [60,61].”

Response to Reviewer 2 Comments

1. Summary

Thank you for consideration of our manuscript. We appreciate the time and effort the reviewer has dedicated to providing insightful feedback on ways to strengthen our paper. Thus, with great pleasure we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and responses we provide below satisfactorily address all the issues and concerns the reviewer has noted.

2. Questions for General Evaluation

Reviewer’s Evaluation

Does the introduction provide sufficient background and include all relevant references?

Must be improved

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Must be improved

Are all figures and tables clear and well-presented?

Must be improved

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

In this article, the authors aim to identify, using hyperimmune sera from mice, rabbits and hamsters, potential regions of structural or sequential homology between the SARS-CoV-2 N protein and the identified human self-antigens.

Comments 1: This purpose should be better described in the abstract to stress well what the authors want to say.

Response 1: We thank the reviewer for the opportunity to make our manuscript even more informative. We agree with this comment, and therefore, we have added the study purpose description in the Abstract: “In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits and hamsters”.

Comments 2: The paper, the authors should be specify why these particular cell lines were chosen and why sera from three different species (mouse, rabbit, and hamster) were included.

Response 2: We thank the reviewer for this valuable note. We selected these particular cell lines because of their human origin, as well as because they belong to multiple tissues, primarily epithelial. It is important to study multiple cell lines since different tissues may have different levels of expression of host cell proteins, and here we primarily focused on cells from epithelial tissues because they are major ports for viral entry. As for the animal models used in our study – we used mice, hamsters and rabbits as standard model animals appropriate for virological and serological studies. Therefore, we have explained the selection of animal sera donors and cell lines in the 2.1 subsection.

Comments 3: They must also describe the positive and negative controls well, in this regard what is written in lines 85-87 is unclear. And also the NCL2, 5, 7 and 10 serums (line 119 -120) need to be better described and/or a reference should be added.

Response 3: We fully agree with this comment. Anti-N monoclonal antibodies NCL2 and NCL10 served as the positive controls, and naïve sera as well as anti-IFNα monoclonal antibodies were used as the negative controls. Therefore, the description of the negative controls, as well as of NCL2 and NCL10 mAbs has been specified in the indicated lines of the manuscript.

Comments 4: The authors must rearrange figure 1, putting the gel first, then the negative anti-IFNa control (explaining why this control is used) and then in sequence negative and positive control of each species  (mouse, rabbit, and hamster). In figure 1, the authors must put under each gel/blot not simply the letter but also the type of serum that was used (obviously only for blots).

Response 4: We thank the reviewer for this note. To clear the description of results, the Figure 1 has been rearranged, and the capture has been specified.

Comments 5: I would also like to understand why have not 8 lanes been made for the gel and for the blots?

Response 5: Many thanks for the comment. We just used a number of wells sufficient for electrophoretic/WB analysis of all samples studied. The eighth line was used for molecular weight marker, and was not counted on gels/blots.

Comments 6: Why have protein extracts not been quantified and normalized by protein content?

Response 6: We thank the reviewer for noting this. In addition to the protein component, lysates contain other molecules that interfere with accurate determination of the protein concentration. Therefore, instead of normalizing by protein content, the samples were normalized by cell count (1 x 106). We have stated this point in the 2.2 subsection: “For each cell line, lysis of 106 cells in PBS was performed...”

Comments 7: Considering that we are talking about a vaccination, where the antibody response is most likely very strong, because the authors think of an ADE mechanism. Are there any studies on other vaccines as well? This topic should be moved to the introduction.

Response 7: Thanks a lot for raising an interesting question. The known examples of ADE provoked by other vaccines have been given in the Discussion section (not in the Introduction to avoid redundant description of ADE in this section): “The cases of ADE development have been reliably shown as a result of the use of vaccines against dengue and Zika fevers [33-36] and respiratory syncytial infection [37]”.

Comments 8: Regarding the ARRIVE guidelines:

• point 2b: It is not specified that Test Statistic was used to calculate the minimum number of animals for the study 
• point 8a: Add the age of the animals
• point 8b: specify the company that supplied the animals
• point 12b: the authors must specify this in the introduction
• point 19: At the end of the article a protocol is specified, why is it not written here too?

Response 8: We thank the reviewer for these important notes. As we are unable to modify the ARRIVE checklist, we have checked that the indicated features are given in the 2.1 subsection of the manuscript.

Comments 9: The article must be thoroughly reviewed carefully, moreover as additional files it would be right to put the blots of all the animals in order to make it clear if the animals have all had the same 'reactivity'.

Response 9: We thank the reviewer for this comment. We would like to draw the reviewer's attention to the fact that the membranes were treated with pooled sera; raw photographs are attached in the Supplement. As suggested, the manuscript has been thoroughly reviewed.

Response to Reviewer 3 Comments

1. Summary

We would like to thank the reviewer for the positive feedback, valuable comments and opportunity to resubmit a revised copy of this manuscript. The point-by-point details of the revisions to the manuscript and our responses to the referee’s comments are listed below. We very much hope that the revised manuscript will be suitable for publication.

2. Questions for General Evaluation

Reviewer’s Evaluation

Does the introduction provide sufficient background and include all relevant references?

Must be improved

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Must be improved

Are all figures and tables clear and well-presented?

Must be improved

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

The manuscript, "Antibodies against the SARS-CoV-2 nucleocapsid protein possess autoimmune properties," was automatically annotated in the context of combating SARS-CoV-2 N and its potential role in post-infectious complications and in the study of new assays. The authors used a combination of Western blots, MALDI-TOF, and bioinformatic structural and conductance analyses. The results demonstrate anti-N reactivity to various antigens, particularly heat, histones, and metabolic enzymes.

The topic is current and active – the issue of autoimmunity and the safety of the N antigen is paramount in the post-COVID context and the subsequent effects of N antigen vaccines. The article has cognitive value and predictive hypotheses that can be tested in cancer research and the development of autoimmune solutions in convalescent patients.

The results are interesting, but the interpretations are too broad or drawn from the data. The manuscript requires several additions and clarifications.

Response 1: We thank the reviewer for careful and thorough evaluation of our work. We revised this manuscript based on your valuable comments.

SCIENTIFIC AND CLINICAL IMPORTANCE

Key significance of the problem: possible autoimmunity induced by anti-N, which is of significant importance for physician safety and the cause of post-COVID disease.

The use of multi-step analysis combining Western blots with MALDI-TOF identification and structural analysis provides a multidimensional picture of reactivity. The authors were able to identify autoreactive targets (HSP70/90, HSP60, histones, PKM, G6PD), which provides a good basis for further research. Anti-NP (influenza) sera are an interesting source as a negative control.

Comments 1: Lack of functional analysis of autoreactivity - It has not been demonstrated whether the observed reactivity in the Western blot translates to actual autoimmune processes in vivo (complementation, ADCC, internalization).

Response 1: We thank the reviewer for this important critique. Indeed, we did not perform functional analyses of autoreactivity, e.g. when uninfected human cell lysates are used as coating antigens in ADCC/CDC assays. However, using SARS-CoV-2 infected cells or recombinant N protein, we earlier demonstrated the ability of anti-N antibodies to provoke CDC and ADCC activity [20], suggesting that anti-N antibodies that bind to the human autoantigens can also provoke the same functional activity. Nevertheless, this hypothesis needs confirmation, which was mentioned in the Discussion section: “We also did not perform functional analyses of autoreactivity, e.g. when uninfected hu-man cell lysates are used as coating antigens in ADCC/CDC assays. However, using SARS-CoV-2 infected cells or recombinant N protein, we previously demonstrated the capacity of anti-N antibodies to induce CDC and ADCC activity [20], suggesting that anti-N antibodies binding to human autoantigens can also elicit similar functional activity”.

Comments 2: Limitations of 1D Western Blots

– The authors note that the 1D SDS-PAGE method does not allow for separation of the device from the system. Western blot 1 does not allow for the detection of bands of very similar mass (e.g., histones H2B/H3) during recording. Combining with MALDI-TOF helps, but requires additional confirmation. It can be recorded if autoreactive labeling is incorrectly removed.

– The lack of densitometry density data for Western blots (the module is not included) and the lack of information on experimental reproducibility (n, number of replicates) are limiting factors.

Response 2: Thank you very much for this note. We are fully aware of the imperfection of 1D SDS-PAGE and have indicated this shortcoming of the work in the study limitations: “First of all, in this study we used the one-dimensional discontinuous SDS-PAGE method, which does not allow for the precise separation of proteins with similar molecular masses”. Here, we present the raw results of identified mixtures of autoantigens with similar mass, and future confirmation using 2D SDS-PAGE can be performed independently by different groups of investigators.

Comments 3: Lack of validation against purified recombinant human samples – To begin with, this paper lacks validation. One Western blot was presented for correlation with scientific data, but without validation using recombinant human samples, which are inaccessible to users. These interactions have been discussed. The authors themselves mentioned the limitations of 1D SDS-PAGE, which further requires validation. There is no specific validation of the conclusions about residual autoimmunity against pure antigens. Please provide validation or amend the manuscript text.

Response 3: We thank the reviewer for this note. We would like to draw the reviewer's attention to the fact that our study is screening, and we have identified a set of potential autoantigens in various cell cultures using Western blotting and mass spectrometry. In the future, for each identified protein, it is necessary to conduct additional validation experiments using recombinant proteins and/or suppressing their expression in cells using siRNA and other methods. Since such in-depth studies were not included in the purpose of this screening study, we have added relevant information to the Discussion section, specifically, to the corresponding statements in the study limitations.

Comments 4: Lack of data on affinity and kinetics of binding – Whether binding is strong, weak, or available (e.g., ELISA, SPR) does not work.

Response 4: We fully agree with the comment. However, affinity and kinetics of binding for each autoantigen can be precisely assessed only using recombinant proteins, which is planned for future work. Accordingly, we have included this point in the study limitations as well.

Comments 5: The methodology requires supplementation with the amount of gel loaded per well and immunoblot parameters (immunoconcentration, incubation time).

Response 5: Thanks a lot for an opportunity to improve the representation of our results. We agree with this suggestion and therefore have detailed WB parameters in 2.2 subsection.

Comments 6: The description of the animal immunization lacks data on the final results.

Response 6: Thanks for the note; the final results of immunizations are now described in 2.1 subsection.

Comments 7: Confirmation of the origin: MALDI-TOF, likely LC-MS/MS.

Response 7: We thank the reviewer for noting this. The identification process did not include a chromatographic step, so the term MALDI-TOF is more appropriate in case of our study.

Comments 8: Table 1 is clear, although it is worth considering legally, if banding is used.

Response 8: We thank the reviewer for the positive feedback. We added the footnote to the Table with description of bands: “*the band number is given according to Figure 1A”.

Comments 9: Suggestions of a link to post-COVID neuropathology are speculative – References to occurrence in neural tissue are interesting, but causal conclusions are unproven. There are no additional in vivo data. These theses should be considered hypotheses.

Response 9: We agree with this valuable comment. An indication of the speculative nature of the assumptions regarding the association of post-COVID syndrome (and, in particular, of persistence of anti-N antibodies) with neuropathology progression has been added to the Discussion.

Comments 10: Lack of comparative data for humans – The authors mention animals between animal models but do not provide patient sera as a reference point.

Response 10: We thank the reviewer for this important note. It should be noted that a similar analysis of the autoreactivity of anti-N antibodies generated in humans as a result of vaccination or infection requires a reliable negative control (naive serum). Given the persistent prevalence of seasonal coronaviruses (including those from before 2019) in the human population, rather than SARS-CoV-2, obtaining such samples is virtually impossible. We previously demonstrated that anti-N antibodies produced against the SARS-CoV-2 antigen are capable of recognizing the N protein of the seasonal coronavirus OC43 [10.3390/v15010230]. Thus, unlike human sera, animal models provide an opportunity to reliably test the affinity of anti-N antibodies for components of the human proteome. However, we have noted that the analysis was performed on animal sera, as a limitation of the study.

Comments 11: Insufficient discussion of possible cross-reactivity unrelated to autoimmunity (so-called sticky antibodies). The aspect of nonspecific reactivity is insufficiently discussed.

– The N antigen is rich in regional basic and disordered proteins – this may generate nonspecific significance.

– The N protein is highly basic and partially disordered – which favors nonspecific binding. This possibility is insufficiently discussed.

Response 11: We thank the reviewer for noting this. The possible explanation of the anti-N antibodies cross-reactivity regardless of the autoimmune reactions has been added to the Discussion.

Comments 12: The introduction is too long – the ADE description could be shortened, focusing on the key points of the paper.

Response 12: As suggested by the reviewer, the Introduction has been modified and focused on the study aim.

Comments 13: Various tables are difficult to interpret – the most important Align/Super rankings explain the explanations for readers outside of bioinformatics. They require access to the method and interpretation.

Response 13: We thank the reviewer for the comment. The rankings presented in Tables 2 and 3 reflect only the degree of affinity between the structure or sequence of the N antigen and the identified human protein. A detailed description of the Align/Super methods, accessible to a wide range of readers and requiring no bioinformatics skills, is presented in the Supplementary Material.

Comments 14: There is no clear summary of practical implications for vaccine design.

Response 14: Thanks for the note; the summary of practical implications of vaccine development has been included in the Conclusions.

Comments 15: The discussion requires a clear distinction between experimental observations and theoretical hypotheses. There is a very extensive section on ADE, but little space is devoted to mechanisms of potential autoimmunity (e.g., molecular mimicry, epitope spreading).

Response 15: Thanks a lot for raising an interesting question. Alternative mechanisms other than ADE that may mediate autoimmune responses have been mentioned in the Discussion.

Comments 16: There is a lack of a thorough discussion of limitations, for example, regarding the possibility of "stickiness" of anti-N antibodies under denaturing conditions.

Response 16: Thanks for the fair comment. This point has been added to the study limitations.

Comments 17: Add validation of key antigens using recombinant human proteins (e.g., HSP90, histone H3, PKM).

Response 17: Thank you for pointing this out. As stated above, the recombinant human antigens were unavailable for us and so validation has not been performed. As suggested by the reviewer, the manuscript text have been modified, specifically by the corresponding statements in the study limitations.

Comments 18: Refine the bioinformatics analysis – indicate the length of the homology regions.

Response 18: We agree with the feasibility of this clarification, and so have added the range of numbers of homologous amino acid residues in 3.3 subsection. The number of homologous amino acid residues for all polypeptide chains of each identified protein is reflected in the Score values and presented in the Supplementary material.

Comments 19: Introduce a quantitative assessment of Western blot signals.

Response 19: We agree with this comment. Indeed, densitometric analysis of the blots could provide some additional information on the content of each protein/band relative to the other proteins within the sample or between samples on the same blot. However, the most informative comparison would be when anti-N sera is compared with the naïve or biological control sera, but these bands come from different blots and direct comparison would not be correct enough. We want to emphasize that our study is more qualitative rather than quantitative. Nevertheless, we have included this point in the study limitations: “Furthermore, since this paper reports on the findings of preliminary experiments, we did not perform WB densitometry or investigate the ability of autoreactive anti-N antibodies of various origins to bind to the purified or recombinant human-derived proteins. Consequently, the results have not undergone validation.”

Comments 20: Limit or mitigate speculation regarding neurological pathogenesis.

Response 20: As suggested, the speculation on the ability of anti-N antibodies to provoke neurological pathologies has been added to the Discussion.

Comments 21: Expand the discussion to include alternative explanations for nonspecific reactivity.

Response 21: We agree with this valuable comment.

Comments 22: Shorten the introduction (ADE in a more concise form).

Response 22: Thanks a lot for the note. The Introduction has been modified accordingly.

Comments 23: Organize Tables 2 and 3 and explain the interpretation of the "Combined norm score."

Response 23: Many thanks for the comment. The data presented in Tables 2 and 3 have been explained in the captions.

Comments 24: Clarify the material and methods (quantities, concentrations, controls).

Response 24: We thank the reviewer for the note. The Materials and methods section has been detailed.

Comments 25: Add information on statistics and the number of replicates.

Response 25: In accordance to the comment, the information on the replicate numbers has been provided in 2.2 subsection. The WB data obtained in the work were qualitative and therefore were not subject to statistical processing.

Comments 26: Lines 14–15 – The phrase "autoimmune reactions triggered by antiviral antibodies" is too general. Please clarify that this is a hypothesis, not a proven mechanism.

Response 26: Thank you for the suggestion. The indicated sentence has been fixed.

Comments 27: Lines 27–28 – Explain why only hamster anti-N antibodies did not show nonspecific cross-reactivity with proteomic extracts from hamsters or humans. This may be due to differences in immunogenicity, not necessarily "lack of autoreactivity."

Response 27: Many thanks for the comment. As suggested by the reviewer, this text fragment has been modified.

Comments 28: Lines 42–49: This fragment is very general. I propose shortening the description of the diversity of SARS-CoV-2 variants.

Response 28: Thanks for the fair comment. The paragraph descripts not the SARS-CoV-2 variants, but N conservatism and clinical implications. It is important for the understanding of the N protein properties and given in a maximally concise form, so we would not like to modify this text fragment.

Comments 29: Lines 64–72: It is worth explicitly mentioning that the presence of the N protein on the cell surface has been a subject of discussion and is not always consistently demonstrated.

Response 29: Thanks a lot for the note. This paragraph has been modified accordingly.

Comments 30: Lines 73–84: The section on ADE is long and deviates from the main topic. I propose shortening it and partially moving it to the discussion section.

Response 30: Thank you for pointing this out. The indicated text fragment is devoted to the protective role of anti-N antibodies instead of ADE triggering, so we consider it as an important part of Introduction.

Comments 31: Lines 101–115: Data on final antibody concentrations are missing (the ELISA titer is not sufficient for interspecies comparison).

Response 31: We appreciate this comment. It should be noted that the study used sera instead of purified antibodies, and sera immunity was standardly assessed by titers. The concentrations of used mAbs are indicated in 2.2 subsection.

Comments 32: Lines 121–129: Please indicate how many μg of protein were loaded per SDS-PAGE lane.

Response 32: Thanks for the fair comment. We have added the protein load values in 2.2 subsection.

Comments 33: Lines 141–158: MALDI-TOF is appropriate, but I suggest adding information on Mascot scoring (CI%, coverage). Please indicate whether an internal control or standard protein was used.

Response 33: Many thanks for the note. The description of MALDI-TOF analysis has been detailed. In Materials and methods section, we have added the data on internal standard used: the spectra were calibrated based on ions, corresponding to trypsin autoproteolysis. We have also added the information about mass spectrometry identification results. All the proteins were reliably identified, as the scores excessed the threshold value. The detailed information is now presented at Supplementary Table S1, namely protein names, scores, intensity coverages and sequence coverages.

Comments 34: Lines 192–202: The authors describe an "identical pattern," but densitometry and independent replicates were not presented. Please provide n.

Response 34: Thanks a lot for the note. The identical banding patterns were observed as a result of analysis of three similar lyzates by WB (n=3), and representative membrane photos are given in the manuscript. The required corrections have been introduced in the sentence.

Comments 35: Lines 218–236: Table 1 is valuable, but requires clarification as to whether the identification was for a single band or a mixture of proteins (which is possible with 1D SDS-PAGE).

Response 35: Thank you for pointing this out. The identification was conducted for the excised bands corresponding to the autoreactive WB zones. These details have been added to the indicated text fragment.

Comments 36: Lines 239–261: The alignment results do not show the lengths of the homologous regions. Please add: was the high match due to short basic motifs or larger domains?

Response 36: Thank you for the suggestion. The information on the major contribution of short, basic or helix-rich motifs to the homology between N protein and human antigens is given in the indicated text fragment.

Comments 37: Lines 263–288: The ADE discussion is interesting, but distracts from the main topic. Please shorten it by ~30–40%.

Response 37: We thank the reviewer for this important note, but believe that a detailed ADE discussion as the most probable and studied mechanism of the development of the anti-N IgG detrimental effects is very important. As suggested before, to balance the discussion, we have extended this text fragment with a mention of other possible non-ADE mechanisms for the development of autoimmune pathologies.

Comments 38: Lines 307–311: Reference to neuropathology requires either citations confirming auto-reactivity to neurons or a significant softening of the wording.

Response 38: We agree with this comment. The statement on the potential ability of anti-N antibodies to provoke neuropathology has been softened.

Comments 39: Lines 319–325: Please include in your conclusions a disclaimer stating that the paper does not confirm autoimmunity in vivo, but only demonstrates the potential for auto-reactivity in vitro.

Response 39: Thanks a lot for the note. A disclaimer has been introduced in Conclusions.

Comments 40: Information about randomization is missing (ARRIVE item 4). Authors marked as N/A - requires justification, which is not provided in the manuscript. No information on blinding during the experimental procedure (ARRIVE item 5). No experimental device (ARRIVE 1b):

- Please indicate whether animals were randomly assigned to groups or provide a justification for the lack of randomization.

- Requires who was aware of the group assignment: immunization, collection, Western blots, and analysis results.

- Specify whether the experimental product is a single individual or, for example, a pooled sample from several animals.

Response 40: Many thanks for the comment. The data on animal randomization, group assignment and experimental product have been added to 2.1 subsection.

Comments 41: The manuscript does not specify animal inclusion/exclusion criteria or graphical criteria (ARRIVE 3a–b). - - - Please indicate whether animal screening or analysis control was performed, and based on the application. If no exclusion occurred, this should be clearly stated.

Response 41: Thank you for pointing this out. Animal inclusion/exclusion criteria have been included in 2.1 subsection.

Comments 42: No statistical calculations of animals and the number of ARRIVE 2b numbers. - Please provide a justification for the number of animals selected, e.g., thermal calculation power. If such calculations are not included, please provide a justification.

Response 42: Many thanks for the comment. A justification for the number of animals selected has been provided in 2.1 subsection.

Comments 43: No description of the animals' health status, pathogen-free status, etc. (ARRIVAL 8b). Please provide information on the animals' health status (e.g., SPF), their management, and whether they have previously undergone any procedures.

Response 43: We agree with this comment. The information on the animals' health status and previous studies has been added to 2.1 subsection.

Comments 44: Details of the procedure (Arrival 9): Please provide information on: antigen transfer (i.m., sc., i.p.), discontinuation of injections, duration and schedule of the procedure, and any acclimation time before the experiment.

Response 44: Thanks a lot for the note. The information on administration route, immunization schedule and acclimation time before the experiment has been added to 2.1 subsection of manuscript.

Comments 45: Husbandry conditions (ARRIVAL 15): Please specify: cage type, bedding type, light-dark cycles, temperature, vehicles, and type of supplementary environmental measures, if any.

Response 45: In accordance to the note, the cage type, bedding type, temperature and light-dark cycles have been specified in 2.1 subsection.

Comments 46: There is no detailed description of pain monitoring and emergency events (ARRIVE 16a-c). Please provide information on:

- method of monitoring animals after immunization,

- use of analgesia (or justification for its absence),

- frequency of welfare checks,

- adverse effects,

- humane endpoints (and whether any have been developed).

Response 46: Thank you for pointing this out. The methods of monitoring, analgesia and humane removal from the experiment are now described in 2.1 subsection.

Comments 47: Pain relief methods. The use of ether anesthesia is outdated in many countries and is not recommended. Ether can cause stress and respiratory irritation.

- Please indicate whether ether is available as an anesthetic agent that complies with regulatory regulations and institutional guidelines.

- Please explain the reason for choosing ether over a more modern method (e.g., isoflurane).

Response 47: Thanks a lot for the note. The justification of ether use has been added to the 2.1 subsection.

Comments 48: Ethics Committee Decision (ARRIVE 14): Please provide the ethics diet, decision/consent number, and ceremony. If consent was not required, please provide justification.

Response 48: Many thanks for the comment. The number and date of the ethical approval are now indicated in 2.1 subsection and Institutional Review Board Statement.

Comments 49: Replacement. The authors used animals only where the humoral response study required a live model. It is not stated whether alternatives were proposed (e.g., in vitro B cell stimulation, immunized splenocytes). Please consider alternative care options.

Response 49: We thank the reviewer for this important note. The inapplicability of alternative options has been mentioned in 2.1 subsection.

Comments 50: Reduction. The authors declare minimal animal management. No statistical calculations were made to justify the number of animals (power analysis). No information on randomization of groups is available, which limits the use of the number. - Please provide a justification for the number of animals selected, e.g., utilization of the analysis's computational power. If such calculations are not included, please provide a justification. Please provide a methodological justification for the reliability of the results in the experiment.

Response 50: Thanks for the fair comment. A justification for the number of animals selected has been given in 2.1 subsection.

Comments 51: Refinement. Free access to food and water was provided. Procedures were performed by qualified personnel. No information is provided on additional analgesia, stress, or pain monitoring. Human endpoints are not described. Please provide full details of any refinement measures, such as analgesia, monitoring frequency, or environmental enrichment.

Response 51: We agree with this comment. The details on monitoring, analgesia and humane removal from the experiment are now given in 2.1 subsection.

Comments 52: Statistical Methods (ARRIVE 7): Please provide a description of the statistical methods used.

Response 52: Thanks a lot for the note. The information on the replicate numbers has been provided in 2.2 subsection. The WB data obtained in the work were qualitative and therefore were not subject to statistical processing.

Comments 53: Exact n values ​​(ARRIVE 3c, 10): For each figure, table, or experiment, the number of animals or samples used in the analysis should be reported.

Response 53: Thank you for pointing this out. In our work, animals were used only to obtain hyperimmune sera, and the number (n) for each species is given in 2.1 subsection.

Comments 54: No serious ethical violations were identified, but this manuscript does not fully meet the ARRIVE 2.0 reporting standards. To ensure full transparency and consistency, authors should revise the manuscript according to the comments.

Response 54: We thank the reviewer for this important note. The manuscript has been revised according to the comments to meet the ARRIVE 2.0 reporting standards.