Advances of the “Miracle Protein” Against Viral Diseases: Lactoferrin in Clinical Trials

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled „Advances of the “miracle protein” against viral diseases: Lactoferrin in clinical trials“ by Ramirez-Rico et al. provides an overview on the outcomes of clinical trials using lactoferrin to combat viral infections. The topic is interesting and of relevance.

 

However, there are quality issues with the text, which needs extensive revisions. The manuscript should undergo language editing and there are several incomplete sentences.

 

The introduction requires major revision. The authors refer to numbers concerning antimicrobial resistance reported by the WHO. There should be a reference to this report. The emerging antimicrobial resistance is mainly because of excessive use of antibiotics to treat bacterial and not viral infections.

The statement of an excessive and inappropriate use of antivirals should be also revised and supported by current literature. The term “inappropriate” is not correct, as medications to treat chronic viral infection are required for the survival of patients (e.g. HIV) or antivirals are applied to immunocompromised patients where re-activation of viruses (e.g. herpes viruses) occurs which can lead to life-threatening disease. During the SARS-CoV-2 pandemic, antivirals were urgently required for treatment of patients.

In general, most viral infections cannot be treated with antivirals, as there is a lack of broadly-acting agents. Therefore, the authors should provide a short summary of the literature on currently available antivirals and where development of antiviral resistance occurs (e.g. HIV).

 

Section 2. Lactoferrin: the authors should mention that Lf is a basic glycoprotein.

 

Figure 1: an explanation of the model should be provided, e.g. location of carbohydrates and ion could be indicated in the figure.

 

Section 2: The authors mention lactoferricin and lactoferrampin. Both are peptides and have antibacterial and antiviral activities. There should be a more detailed literature summary about the antiviral activities of these short peptides.

 

Paragraph (lines 120-136) needs text revision.

 

Section 5. Lines 185 – 194 should be moved to section 5.1. adenoviruses. Disease (line 186) should be changed to infection since it was an infection experiment with HEp-2 cells.

 

5.2. Influenza A. Avian influenza is a problem, but seasonal influenza is the major health problem, this should be mentioned.

 

6.2. Gastroenteritis: it should be mentioned which viruses cause gastroenteritis. In line 277 sotavirus should be replaced by rotavirus. In line 310 the authors refer to a study with astroviruses, but mention this in the paragraph about noroviruses.

 

Line 339: Moriuchi and Moriuchi

 

Also, there are incomplete sentences in the Table.

“Cold” should be replaced by “Common cold”.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript provides a thorough and well-structured overview of the antiviral properties of lactoferrin (Lf), integrating mechanistic insights with evidence from clinical studies across a wide spectrum of viral infections, including hepatitis, influenza, rotavirus, norovirus, poliovirus vaccination, and COVID-19. Although the antiviral activity of lactoferrin has been discussed in previous narrative and systematic reviews, particularly during the COVID-19 era, the present work is valuable in that it consolidates diverse experimental and clinical findings into a single, accessible synthesis. A notable strength of the review is its broad coverage of viral pathogens, the inclusion of clinical trial data, and the consideration of different formulations, including liposomal lactoferrin, and immunoglobulin-rich fractions. The scientific soundness of the manuscript can be considered moderate, though somewhat inconsistent. On the positive side, the review accurately describes established antiviral mechanisms of lactoferrin, including its interaction with heparan sulfate proteoglycans and its immunomodulatory effects, appropriately acknowledges the conflicting results of COVID-19 clinical trials, and recognizes potential limitations related to bioavailability. However, there is limited critical evaluation of the methodological quality of the cited clinical trials, including aspects such as randomization, blinding, sample size, and endpoint selection, which constrains the overall rigor of the analysis and of the review as a whole. The figures and table presented in the manuscript are generally appropriate and well aligned with the main text. Unfortunately, in its current version, the review contains numerous sections that are difficult to read and understand. I believe the manuscript may be suitable for publication after appropriate revisions and satisfactory responses to the comments below.

Major Comments:

Lines 33-39. This paragraph conflates antimicrobial resistance and antiviral resistance. The statement “antimicrobial resistance causes approximately 700,000 deaths worldwide each year” presents data on deaths caused by antimicrobial resistance, but it is unclear what proportion of these deaths is attributable specifically to antiviral resistance. Please clearly distinguish between antimicrobial resistance and antiviral resistance. Update the economic data and provide appropriate references specifically related to antiviral resistance.

Lines 45-46. “Antiviral resistance is defined as the ability of viruses to mutate and replicate in the presence of an antiviral drug [2].” – Please verify the accuracy of this definition, as antiviral resistance is generally understood as the ability of viruses to withstand the effects of drugs that were previously effective against them.

Line 85. “cross the nucleus” – Since the text refers to a protein, the more appropriate term is “translocate to the nucleus”.

Lines 162-163. “It has been shown that the effect of iron-free apo-Lf on some viruses was greater than that of holo-Lf [36]” – Please provide a more appropriate reference and specify which viruses are meant instead of “some viruses,” as the cited work does not clearly present this information.

Lines 173-178. The paragraph on breastfeeding does not include references supporting the statement that Lf ensures a “smaller number of infectious diseases caused by respiratory syncytial virus, rotavirus, or vesicular stomatitis virus [40,41].” References 40 and 41 do not appear to be appropriate or directly related to the role of lactoferrin.

Lines 278-279. “apo-Lf displayed a substantially higher selectivity index, about 600-fold greater” – Please clarify how the selectivity index was calculated (e.g., CC50/IC50) for transparency.

Lines 211-218. “In 2010, Taha et al. tested different whey proteins against avian influenza A (a-lactalbumin, b-lactoglobulin, and Lf) in MDCK cells.” – Please provide the reference immediately after this statement. It appears that reference [50] is intended, but references [48,49] follow instead, which disrupts the citation sequence and creates confusion.

Lines 345-347. “The most important study of Lf against a viral agent targets the causal agent of the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is caused by the etiological agent coronavirus 2019 (COVID-19)” – This sentence is confusingly formulated, and “coronavirus 2019 (COVID-19)” refers to the disease, not the etiological agent. Please revise for clarity and scientific accuracy.

Lines 367-369. “The efficacy lies in Lf's serving as an angiotensin-converting enzyme 2 (ACE2) receptor blocker that prevents binding of the viral spike protein S to the host cell, thereby blocking viral fusion with the cell membrane [80,81].” − The available evidence does not support lactoferrin as a true ACE2 receptor antagonist. References [80,81] also do not substantiate the claim that efficacy lies in ACE2 receptor blockade. Please review this section, correct the text accordingly, and provide references that are directly relevant to the stated mechanism.

Author Response

 "Please see the attachment."

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully revised their original manuscript, so that it is now significantly improved. In my opinion, the manuscript is suitable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed the authors’ response and the revisions made in the latest version of the manuscript. In my opinion, the authors have appropriately addressed the comments and have substantially improved both the clarity of the text and the presentation of the data. The cited literature has also been carefully revised and corrected. Overall, in its current form, the manuscript can be recommended for publication.