Potential Diagnostic and Research Applications of a Recombinant Antibody Directed Against Ferrated Triacetylfusarinine C from Aspergillus fumigatus

Round 1

Reviewer 1 Report

1. For the ELISA assay development, FeDAFC-BSA is used as the coating antigen, while FeTAFC is the targetbeing detected. Although DAFC is structurally related to TAFC, the two molecules are not identical. It would be helpful for the authors to clarify whether the antibody binding affinity to FeDAFC and FeTAFC has been directly compared.
2. In Table 1, the nominal concentration of 63 ng/mL yielded a mean measured concentration of 197.1 ng/mL, indicating a large discrepancy between the expected and measured values. However, the manuscript mainly discusses the %CV and does not address the accuracy or recovery of the assay.
3. The proposed diagnostic application of the assay would ideally be supported by testing urine samples from patients with invasive aspergillosis. In the current study, the assay was evaluated only using urine samples from healthy donors and spike-in experiments with FeTAFC standards. While these experiments demonstrate that the assay can function in a urine matrix, they do not demonstrate that endogenous FeTAFC produced during infection can be reliably detected.Inclusion of clinical specimens from patients with invasive aspergillosis, as well as from individuals with other infections, would significantly strengthen the clinical relevance of the study.
4. The manuscript reports measurable FeTAFC levels in urine samples from healthy volunteers. Given that TAFC is a siderophore typically produced by Aspergillus species during infection, this observation is somewhat unexpected. The authors should clarify whether these signals represent true FeTAFC detection, assay background, or potential cross-reactivity. Confirmation using an orthogonal analytical method such as LC–MS would increase confidence in the specificity of the assay. In addition, evaluating samples from other fungal infections would help demonstrate the diagnostic specificity of FeTAFC in clinically relevant settings.
5. The authors report decreased growth at antibody concentrations of 40 and 100 pmol/disk,butthe comparisons with the PBS control yield p-values of 0.7908 and 0.2720, respectively, indicating that these differences are not statistically significant. The current data do not demonstrate a clear dose dependent inhibitory effect of the antibody on fungal growth in the wildtype strain.

1. The title of Table 1 refers to “reproducibility”, whereas the data presented correspond more closely to intra-assay precision (%CV). Clarifying this terminology would improve accuracy.
2. Consistent terminology throughout the manuscript would improve clarity.(e.g. FeTAFC ELISA, TAFC ELISA, and FeTAFC immunoassay)
3. The statistical tests used for the growth inhibition experiments should be clearly stated in the Methods section.
4. Some units are inconsistently formatted (e.g., line 134: ng/mL vs line 413: ng/ml).
5. The font type and font size used in the figures appear inconsistent. Standardizing the formatting across figures would improve the overall presentation of the manuscript.The resolution and clarity of someimages should be enhanced to ensure that labels and graphical elements are clearly visible.(e.g.Fig.2c-e)

Author Response

Please see attachment.

Changes are marked in yellow highlight in attached R1 manuscript.

Manuscript is also provided with unhighlighted changes.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The article aims to demonstrate the potential usefulness of monoclonal antibodies anti- ferrated triacetylfusarinine C in the diagnosis and treatment of aspergillosis.

The authors constructed an ELISA test for the detection of FeTAFC antigen using specific monoclonal antibodies (published in their earlier study). They demonstrated that the sensitivity of the test is sufficient to detect FeTAFC in urine samples. The developed test was calibrated using FeTAFC diluted in both buffer and human urine samples, however SD was quit high. The further evaluation is required, and it is unfortunate that no trial tests were performed on samples from patients with/without aspergillosis. I believe that this is the most important limitation of this study.

As second part, a series of experiments were conducted to examine the effect of recombinant monoclonal anty-FeTAFC IgG on A. fumigatus growth. The inhibitory effect offers the possibility of using them in the treatment of aspergillosis. These results provide a basis for planning future studies, e.g. on animal models.

38 „a mortality rate of between 30-90 % depending on the underlying condition” - Mortality also depends on early and appropriate treatment, including access to triazoles or amphotericin B.

Were is Figure 1b?

What means „%CV"? coefficient of variation? How was calculated?

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Authors,

Aspergillosis, caused by various species of the genus Aspergillus, is one of the most common fungal infections worldwide, affecting both immunocompetent and immunocompromised individuals. Of those species, A. fumigatus is the leading cause of disease, due to its widespread presence in all types of environments, the size of its conidia, and the presence of melanin in its cell wall that protects it from the host’s innate immune response. A wide variety of alternatives exist for aspergillosis diagnosis; however, there is no single method that is useful in all suspected cases. While those procedures have demonstrated diagnostic utility, they also present limitations across different clinical contexts and sample types. In addition, the resistance of A. fumigatus to antifungal drugs has led to therapeutic failures, representing another global problem. With this information in mind, I have reviewed the manuscript entitled “Analytical and Potential Growth Inhibitory Properties of a Recombinant Antibody Directed Against Ferrated Triacetylfusarinine C from Aspergillus fumigatus” (Manuscript ID jof-4193787) by Dwyer M, et al. In spite of the merits of this study (mainly, the large amount of experimental work that it contains), I have some comments and suggestions for the authors. I am going to mention those comments and suggestions in the same order they appear in the manuscript:

- The title of this manuscript is excessively ambiguous. The diagnostic use of the recombinant antibody, which is what most of the experimental work described is directed towards, is barely represented in that title. The title, as it appears in the manuscript, features the “…Potential Growth Inhibitory Properties of a Recombinant Antibody Directed Against Ferrated Triacetylfusarinine C from Aspergillus fumigatus,” and not its diagnostic use, which is the most extensively addressed. I suggest changing the title to one that makes greater reference to the diagnostic use of the recombinant antibody.

- In Discussion, between line 411 and 414, the authors write “Moreover, the IgG [anti-FeTAFC] was also shown to impede A. fumigatus growth in vitro, and to alter the supernatant FsC/TAFC ratio, under iron-free growth conditions. These data illustrate the potential impact of new concepts to address aspergillosis detection and treatment”. The authors must explain more clearly the manner in which the impediment of A. fumigatus growth in vitro, and the alteration of the supernatant FsC/TAFC ratio, under iron-free growth conditions “impact of new concepts to address aspergillosis detection and treatment,” when diagnosis and treatment are very different events.

- The rigorous execution of the experiments of the analytical phase of the development of “a rapid, specific, and sensitive FeTAFC ELISA for the detection of FeTAFC in urine using IgG [anti-FeTAFC] to overcome the technological accessibility problems associated with mass spectrometry instrumentation” is the main contribution of this study. Nevertheless, its publication without validation with clinical samples seems somewhat rushed, especially if, as the authors rightly comment, certain components of urine and environmental aspects could interfere with the results.

- In conclusion, between line 542 and 545, the authors write “It is also proposed that the antibody could potentially have some therapeutic applications against A. fumigatus, as it recognises FeTAFC, a siderophore essential for the growth and virulence of A. fumigatus” Certainly, the in vitro demonstration of the possible inhibitory effect of IgG [anti-FeTAFC] on the growth of A. fumigatus is a unique, and too preliminary, proof of the potential therapeutic application of the antibody against A. fumigatus infection. Other proof are necessary. I suggest, therefore, that the experiments related to the therapeutic use of the recombinant antibody not be included in this publication, that other proof be added, and together they form part of another publication.

Dear Authors,

Aspergillosis, caused by various species of the genus Aspergillus, is one of the most common fungal infections worldwide, affecting both immunocompetent and immunocompromised individuals. Of those species, A. fumigatus is the leading cause of disease, due to its widespread presence in all types of environments, the size of its conidia, and the presence of melanin in its cell wall that protects it from the host’s innate immune response. A wide variety of alternatives exist for aspergillosis diagnosis; however, there is no single method that is useful in all suspected cases. While those procedures have demonstrated diagnostic utility, they also present limitations across different clinical contexts and sample types. In addition, the resistance of A. fumigatus to antifungal drugs has led to therapeutic failures, representing another global problem. With this information in mind, I have reviewed the manuscript entitled “Analytical and Potential Growth Inhibitory Properties of a Recombinant Antibody Directed Against Ferrated Triacetylfusarinine C from Aspergillus fumigatus” (Manuscript ID jof-4193787) by Dwyer M, et al. In spite of the merits of this study (mainly, the large amount of experimental work that it contains), I have some comments and suggestions for the authors. I am going to mention those comments and suggestions in the same order they appear in the manuscript:

- The title of this manuscript is excessively ambiguous. The diagnostic use of the recombinant antibody, which is what most of the experimental work described is directed towards, is barely represented in that title. The title, as it appears in the manuscript, features the “…Potential Growth Inhibitory Properties of a Recombinant Antibody Directed Against Ferrated Triacetylfusarinine C from Aspergillus fumigatus,” and not its diagnostic use, which is the most extensively addressed. I suggest changing the title to one that makes greater reference to the diagnostic use of the recombinant antibody.

- In Discussion, between line 411 and 414, the authors write “Moreover, the IgG [anti-FeTAFC] was also shown to impede A. fumigatus growth in vitro, and to alter the supernatant FsC/TAFC ratio, under iron-free growth conditions. These data illustrate the potential impact of new concepts to address aspergillosis detection and treatment”. The authors must explain more clearly the manner in which the impediment of A. fumigatus growth in vitro, and the alteration of the supernatant FsC/TAFC ratio, under iron-free growth conditions “impact of new concepts to address aspergillosis detection and treatment,” when diagnosis and treatment are very different events.

- The rigorous execution of the experiments of the analytical phase of the development of “a rapid, specific, and sensitive FeTAFC ELISA for the detection of FeTAFC in urine using IgG [anti-FeTAFC] to overcome the technological accessibility problems associated with mass spectrometry instrumentation” is the main contribution of this study. Nevertheless, its publication without validation with clinical samples seems somewhat rushed, especially if, as the authors rightly comment, certain components of urine and environmental aspects could interfere with the results.

- In conclusion, between line 542 and 545, the authors write “It is also proposed that the antibody could potentially have some therapeutic applications against A. fumigatus, as it recognises FeTAFC, a siderophore essential for the growth and virulence of A. fumigatus” Certainly, the in vitro demonstration of the possible inhibitory effect of IgG [anti-FeTAFC] on the growth of A. fumigatus is a unique, and too preliminary, proof of the potential therapeutic application of the antibody against A. fumigatus infection. Other proof are necessary. I suggest, therefore, that the experiments related to the therapeutic use of the recombinant antibody not be included in this publication, that other proof be added, and together they form part of another publication.

Author Response

Please see attached file

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

1. The most significant limitation of this study is the absence of validation using clinical samples from patients with invasive aspergillosis. While the assay demonstrates the ability to detect spiked FeTAFC in urine from healthy donors, this does not establish its diagnostic utility in real-world settings. Without testing patient samples, key parameters such as sensitivity, specificity, and clinical relevance cannot be evaluated. 

2. The detection of measurable FeTAFC levels in urine from healthy individuals raises important concerns regarding assay specificity. Although the authors cite previous literature suggesting possible background presence, the current study does not rule out assay cross-reactivity or non-specific signals. Validation using an orthogonal method (e.g., LC–MS) is necessary to confirm that the detected signals indeed represent FeTAFC rather than assay artifacts.

1. Table 1 is now described as “precision” rather than “reproducibility,” which is appropriate. However, the table legend should clearly define all parameters (e.g., CV, concentration levels, replicates).
2. Some figures (e.g., Fig. 2c–e) require improved resolution and consistent font size to enhance readability

Author Response

Please see attached file

 

Author Response File: Author Response.pdf

Reviewer 3 Report

I endorse.

I endorse.

Author Response

Thank you to Reviewer 3 for their endorsement of our work.

Round 3

Reviewer 1 Report

The study presents an interesting approach for detecting FeTAFC using a recombinant antibody-based ELISA. However, the lack of clinical validation and unresolved concerns regarding assay specificity and functional claims significantly limit the impact of the work. Substantial additional validation would be required to support the proposed diagnostic and therapeutic relevance.

The authors have addressed several technical and presentation-related issues adequately. However, the major concerns regarding clinical relevance, assay specificity, and functional conclusions remain insufficiently resolved.

Lack of clinical validation

 No orthogonal validation (LC-MS)

Weak evidence for growth inhibition

 Unresolved assay design concern (FeDAFC vs FeTAFC)

Ｆigures could benefit from improved resolution and clearer presentation. 

Author Response

Reviewer 1:

The study presents an interesting approach for detecting FeTAFC using a recombinant antibody-based ELISA. However, the lack of clinical validation and unresolved concerns regarding assay specificity and functional claims significantly limit the impact of the work. Substantial additional validation would be required to support the proposed diagnostic and therapeutic relevance.

The authors have addressed several technical and presentation-related issues adequately. However, the major concerns regarding clinical relevance, assay specificity, and functional conclusions remain insufficiently resolved.

Lack of clinical validation

 No orthogonal validation (LC-MS)

Weak evidence for growth inhibition

 Unresolved assay design concern (FeDAFC vs FeTAFC)

Author response:

Thank you most sincerely for your overall commentary and expertise. We agree that our work is an interesting approach to FeTAFC detection and that it will require more validation, including the specific points you have illustrated, for human diagnostic application. Nonetheless, as a piece of research, it is unprecedented, and from a FeTAFC detection perspective highlights that FeTAFC can be specifically detected by ELISA compared to, and distinct from, other A. fumigatus siderophores. The use of IgG [anti-FeTAFC] to impede or attenuate A. fumigatus growth is the first suggestion that anti-siderophore antibodies or immunologicals could have a role in suppressing fungal growth by blocking ferrated siderophore uptake.

Detailed comments

Ｆigures could benefit from improved resolution and clearer presentation. 

Author response: 

We have provided a pdf of improved Figures in R2 and requested the journal to insert accordingly into the manuscript as we could not do same without altering overall formatting.