Radioimmunotherapy as a pathogen-agnostic treatment method for opportunistic mucormycosis infections
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Invasive fungal infections (IFIs) such as mucormycosis are causing devastating morbidity and mortality in immunocompromised patients as anti-fungal agents do not work in the setting of a suppressed immune system. The coronavirus disease 2019 (COVID-19) pandemic has created a novel landscape for IFIs in post-pandemic patients, resulting from severe immune suppression caused by COVID-19 infection, comorbidities (diabetes, obesity) and immunosuppressive treatments such as steroids. The antigen–antibody interaction has been employed in radioimmunotherapy (RIT) to deliver lethal doses of ionizing radiation emitted by radionuclides to targeted cells and has demonstrated efficacy in several cancers. One of the advantages of RIT is its independence of the immune status of a host, which is crucial for immunosuppressed post-COVID-19 patients. In the present work we targeted the fungal pan-antigens 1,3-beta-glucan and melanin pigment, which are present in the majority of pathogenic fungi, with RIT, thus making such targeting pathogen-agnostic. We demonstrated in experimental murine mucormycosis in immunocompetent and immunocompromised mice that lutetium-177 ( 177 Lu)-labelled antibodies to these two antigens effectively decreased the fungal burden in major organs, including the brain. These results are encouraging because they show the effectiveness of pathogen-agnostic RIT in significantly decreasing fungal burden in vivo , while they can also potentially be applied to treat the broad range of invasive fungal infections that express the pan-antigens 1,3-beta-glucan or melanin.
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Thank you for addressing the reviewer comments and revising your manuscript accordingly. I am pleased to tell you that your article has now been accepted for publication in Access Microbiology.
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Thank you to the authors for their in-depth revision of the manuscript following reviewer comments. However, some important issues were not satisfactorily addressed and I would like to give you another opportunity to address these final points.
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Comments to Author
Thanks to the authors for their reply and efforts to improve the experimental methodology, however I believe the manuscript still has details to be improved including some mentioned in the original review. 1. Title should reflect this study is focused in mucormycosis 2. Irradiating mice is not a proxy for a COVID-19 model (unless there is evidence showing otherwise) 3. I don't agree with the authors about antifungals not being standard of care in managing mucormycosis and that antifungals do not work in immunosuppressed populations. Antifungals should be the comparative when studying new antifungal molecules 4. There are mislabeled graphics.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure …
Comments to Author
Thanks to the authors for their reply and efforts to improve the experimental methodology, however I believe the manuscript still has details to be improved including some mentioned in the original review. 1. Title should reflect this study is focused in mucormycosis 2. Irradiating mice is not a proxy for a COVID-19 model (unless there is evidence showing otherwise) 3. I don't agree with the authors about antifungals not being standard of care in managing mucormycosis and that antifungals do not work in immunosuppressed populations. Antifungals should be the comparative when studying new antifungal molecules 4. There are mislabeled graphics.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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While the reviewers found this article interesting and of potential importance, they have made suggestions for improvement. We would like you to strongly consider the reviewers' comments and amend your manuscript accordingly. Please pay particular attention to the comments concerning experimental controls and data presentation. Furthermore, please consider revising your title in line with reviewer 2 comments that the title is misleading. Due to the nature of these revisions, please let us know if you need more time to resubmit.
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Comments to Author
This is a study exploring the therapeutic potential of mAbs tagged to melanin and 1-3 beta gluon on a murine model of mucormycosis. The proposal authors do in this manuscript is intriguing, however is lacking rigorous controls and requires essential set of experiments. Here some recommendations and comments: 1. Title: The title is misleading, authors are presenting a murine model of mucormycosis and not for all opportunistic fungal infections. Also, the murine model is not a post-COVID-19 model of mucormycosis. Hence, the title is not correctly reflecting what the manuscript is reporting. 2. Introduction Similar as with the title, the introduction doesn't reflect what the authors did in this project. Also, present wrong terms such as "popularly known as black fungus infection". Authors mention …
Comments to Author
This is a study exploring the therapeutic potential of mAbs tagged to melanin and 1-3 beta gluon on a murine model of mucormycosis. The proposal authors do in this manuscript is intriguing, however is lacking rigorous controls and requires essential set of experiments. Here some recommendations and comments: 1. Title: The title is misleading, authors are presenting a murine model of mucormycosis and not for all opportunistic fungal infections. Also, the murine model is not a post-COVID-19 model of mucormycosis. Hence, the title is not correctly reflecting what the manuscript is reporting. 2. Introduction Similar as with the title, the introduction doesn't reflect what the authors did in this project. Also, present wrong terms such as "popularly known as black fungus infection". Authors mention "some countries" have a high prevalence of Rhizopus species, this needs to be more specific about which countries and also, is this prevalence for Rhizopus species or for Mucromycosis?. Also, concepts expressed in lines from 36 to 42 need to be rephrased as are confusing. 3. Methods and materials Authors don't explain why they chose these fungal targets. It might be understood from one side that these two fungal targets are considered universal in fungi, however it doesn't make sense to test beta-d-glucan as a therapeutic target for mucormycosis as this is one of the fungal infections that will produce minimum or none beta-glucan. In addition, what type of melanin authors used as target? is it a pan-melanin? is it L-dopa or DHN? this is necessary information when talking about targets mainly if the ultimate goal is using these mAbs against all or other fungal infections in future. The in vitro binging assays are informative but also, it would be necessary to also include immunofluorescent images of spores and hyphae. About the Murine model, I wonder if the mice were immunocompromised or modified in any way? otherwise it seems to be a "healthy" non-immunocompromised model of mucormycosis. As mentored above, authors don't mention this model is a post-COVID-19 model, hence this can't be translated to findings in COVID-associated mucormycosis (CAM). About author's methods to measure fungal burden, this is not the standardised and usual way to test it. I fear this methods might not be very reproducible. CFUs or qPCR are the conventional methods to evaluate fungal burden. Another important methodology lacking in this model is histological evaluation of harvested organs and evaluation of histological changes and also fungal burden, necrosis, invasion among other different features important in evaluating therapeutic agents using a murine model. Also, in this model, authors didn't use a standard of care to compare the therapeutic effect, for example L-AMB or isavuconazole. This is a major problem of experiments design. 4. Results, Figure and tables Figure 1 and results described in point 3.1 need statistical information. Also, is interesting that in figure 1.B the Y axis highest value is 0.6 and in the rest of figures is between 2-3. This has two issues 1) misleads the results visually added to the fact that there is not info about mean and SD and p values. 2) same in figure 1B and also 1C, the binding is completely different meaning that probably the binding is higher to spores than is to hyphae. This is why having immunofluorescent microscopy on spores and hyphae will contribute to the knowledge on how these mAbs are working. Figure 5 and plots in it along with the corresponding description of the results lack of statistical information, mean and SD. This info is key as authors claimed that 100 uCi had better effect than using 50 uCi but in plots some of the error bars and means merge.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The work presents a very relevant theme in the current scenario, where the WHO has published a list of priority fungal pathogens for which it is essential to research and develop new therapeutic alternatives, the proposal to use monoclonal antibodies is a very interesting strategy to combat invasive fungal infections, since it thinks beyond the usual pharmacological alternatives. 1. Appropriate and reproducible methodology. 2. Results well described, however images should be improved. Improve the identification of Figure 1 by improving the quality of the font identifying the organ on each plate or improving the legend by adding more information. Enlarge the images of Figure 4 and add to the legend the meaning of the colors presented in the image or highlight in the image the points of interest of the …
Comments to Author
The work presents a very relevant theme in the current scenario, where the WHO has published a list of priority fungal pathogens for which it is essential to research and develop new therapeutic alternatives, the proposal to use monoclonal antibodies is a very interesting strategy to combat invasive fungal infections, since it thinks beyond the usual pharmacological alternatives. 1. Appropriate and reproducible methodology. 2. Results well described, however images should be improved. Improve the identification of Figure 1 by improving the quality of the font identifying the organ on each plate or improving the legend by adding more information. Enlarge the images of Figure 4 and add to the legend the meaning of the colors presented in the image or highlight in the image the points of interest of the result. 3. The discussion should be improved by confronting the data obtained with what is in the literature, being similar works, or works that are about new therapeutic alternatives directed to R. oryzae. Why does this work stand out from the others? What is the role of the use of monoclonal antibodies in the resistance scenario? Are they better than traditional drugs? In which aspects? Enrich the discussion.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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