Trained Immunity as a Prospective Tool against Emerging Respiratory Pathogens
Round 1
Reviewer 1 Report
Trained immunity Joseph.
The author reviews the topic of trained immunity (TI). In general, the manuscript is well-written and organized with high quality illustrations, but superficial in comparison to other similar publications in the field. Indeed, this manuscript reviews prior experimental studies, but does not provide in-depth insights (best TI agents, best procedures, routes, etc.). Moreover, as detailed below, it has significant conceptual shortcomings that evidence the author’s knowledge gaps in the field. Given that there are several recent reviews on this topic, this manuscript adds little to the current literature.
Specific comments:
1) There are trivial mistakes: In line 49, the author refers to BCG as: Bacillus Calmette–Guéri (it should be Guérin) (BCG, a live, attenuated virus originally used against tuberculosis)... Evidently, BCG is an attenuated bacterium derived from Mycobacterium bovis.
2) It would be expected for the author to describe in detail mechanism of training immunity such as what epigenetic programming takes place, what histones are involved, what specific histone modifications may take place and how this reprogramming affects gene expressions at the cytokine level. Instead, the review refers only to these processes in very general terms: “histone methylation and acetylation that confer “easy (?) gene expression and consequent overexpression that elicits antipathogen response.” Is all gene expression upregulated or are there genes that are downregulated as well?
3) The dichotomy between TI and tolerance is not addressed in the review. What factors lead to each of these opposite outcomes?
4) Line 97: to what is the author referring to by “low viremia associated with malarial infections”?
5) Figure 1: Response levels and time frames, as described, are similar by both adaptive immunity and TI, except for a rapid response in TI after primary and secondary challenge. So, what is then the advantage of TI? What does it mean “primary challenge/vaccine/infection” here? With related or unrelated pathogen/both? Succinctly, after for example BCG vaccination (or unrelated infection) and induction of TI, the primary encounter with a pathogen yields an enhanced innate response. Make a diagram compatible with the correct statement made in lines 242-245. Also provide reference as this was envisioned before this review. Training immunity is actually the memory of the innate response, and should not be morphed into innate immunity as it is well- explained in previous reviews by Netea et al. The current manuscript does not make that clear.
6) Line 155: transgenic mice models are mentioned but details should be given. What is the transgene? Moreover, basic transgenic animal models do not fully mimic human infections and more sophisticated models are being developed including specialized humanized mice.
7) Table 1, BCG TI vs Influenza A: that sentence should read BCG enhanced innate immunity (TI) and influenza vaccines increase acquired immunity at higher levels. MMPs: please define acronym after first use.
8) Lines 204-205: Which Covid vaccines? Please, specify.
9) Diagrams in Figure 2: do they refer to actual or planed experiments? If the former, please indicate corresponding citations in the text and figure legends.
10) Lines 275-276: Are there any potential novel trained immunity inducers for the elderly, newborns described in the literature?
11) Lines 279-286. Please, also mention that this type of approach (combining TI with antigen-specific vaccines) has already been proposed and provide appropriate references.
12) Regarding metabolic processes, the onset of glycolysis is important. However, this is not even mentioned in the review.
13) The review describes how TI could bolster acquired antibody responses. What about for the acquired cellular immunity (e.g., T-cell subsets)? For example, MV130 induces Th1, Th17 and IL-10 (see and revise reference 56 to expand).
14) Conclusion: this paragraph is adequate.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
John Joseph offers here a brief synthesis of the works done on trained immunity and respiratory infections. Despite some inaccuracies and non-justified shortcuts pointed below, the review is easy to read and presents nice synthetic figures. However, it is hard to understand from where the author is speaking, why is he interested in respiratory trained immunity and above all with what competences the author emerges in the field of trained immunity? May be a couple of sentences at the beginning of the review explaining the why and how of the interest of the author for this field would be useful.
Line 10: the meaning of the sentence “the vulnerability of current vaccine technologies characterized by a slow onset of action and antigen-specific immune response.” is not clear, how the current vaccine technology can be characterized by antigen specific immune response. All the vaccine technologies are characterized by antigen-specific immune response, since vaccination definition is the triggering of an antigen specific immune response.
Line 27, to our knowledge, none of the citations 1 to 5 tackle the role of γδT cells in innate defense against respiratory pathogens. Please add a reference.
Line 95: “Respiratory epithelial stem cells also featured characteristic innate immune memory by providing enhanced access to chromatin”. This sentence is confusing, epigenetic reprogramming does not consist of just opening the chromatin, to my understanding it both triggers chromatin’s opening of some genes and chromatin’s closing of others. Moreover, in trained cells, once the chromatin is opened at some genes locations, the return to homeostasis does not modify this opening, indeed it is the very basis of imprinting that the epigenetic modifications remain, after the removal of the stimulus. Please rephrase.
Line 100: “epigenetic modifications were observed to exert robust interferon γ (IFNγ) and Th1 cytokine productions”, again to my understanding epigenetic modifications don’t “exert” directly Th1 cytokine productions, they allow the stimulus to trigger this robust production.
Line 112: Ref 41 is not about trained immunity induction by MDP since MDP treatment must be delivered 1 but not 4 days before challenge. Similarly, references 42, 43, 44 are more about direct short term immune stimulation than about trained immunity.
Line 139: whereas BCG-mediated trained immunity has been shown protective for elderly, and especially for respiratory infections Giamarellos-Bourboulis Cell 2020 (doi.org/10.1016/j.cell.2020.08.051)
Line 155: the separation between Figure 1 legend and the main text is not clear.
Line 157: “The current phase of influenza 157 management involves trained innate immunity to recuperate host immune defenses.” What publication can justify this assertion?
Line 158: It is strange to introduce the BCG here whereas you cited it for trained immunity several times before.
Line 182: “orchestrate” seems inappropriate here.
Chapter 3: There are several back and forth from SARS-CoV2 to Influenza infections, and from BCG to MV130, LPS and Zymosan which are difficult to follow.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
1. The review is concisely written, and the need of developing vaccines based on trained immune response as well as the need of hybrid vaccine has been mentioned and supported. The following concerns should be taken care of
Influenza- please avoid repetition- line 50
2. Lines 59-61: it is not clear in these lines whether the authors is talking about the immune response after an infection of after vaccination to attain immunity. Please clearly mention it.
3. Epigenetic alterations are not always reversible, please clear this- is author talking about immune related or disease related.
4. Please cite lines 59-75
5. BCG also induced hetero- line 96- please check
6. with immunosenescence and immunodeficient—line 124- please check
7. section 3, para 2 is too long, please split it in two
8. the murine model-line 191- please be specific to the model used as the immune response and immune make up of mouse and rat differs.
9. Animal word has been used for reference#48 and 49, please specifically mention the animal used- mice or rat?
10. Numerous clinical trials have been conducted in neonates, adults, and the elderly population to investigate the effect of BCG on trained immunity- please cite the recent ones or any article summarizing these.
11. The concept of hybrid vaccine has been introduced at the end of the review; it will be better to discuss in brief in the introduction section also to make the aim of this review clearer
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
The paper was written in an interesting and clear manner. The different sections form a coherent whole. The figures have been carefully prepared and are a valuable addition to the text.
Major note: line 49 - BCG is not a virus!!! Such a mistake is often identified in unprofessional texts in the Internet space, but in a scientific article it should not happen. The reviewer attributes it to "absentmindedness" - otherwise the presence of such a mistake should disqualify the work.
Minor comments:
1/ line 93 - capital letter in the name "toxoplasma"
2/ unify the notation in vivo, in vitro, ex vivo, etc. - italics
3/ Table 1 - expand abbreviations: i.d, i.m etc.
4/ Figure 1 - the graph illustrating the dynamics of antibody appearance should show a more pronounced decrease in antibody levels after the primary response to antigen, as sometimes antibodies disappear almost completely and the information about them (and about the antigen) is stored in memory B cells
Author Response
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Author Response File: 
Author Response.docx
Reviewer 5 Report
Throughout, the report summarizes what has been reported so far.
The figures and tables are also clearly depicted in accordance with the argument.
No major changes are required in this version. However, if you are the sole author, use "I" instead of "we".
Author Response
I thank the reviewer for your valuable time in reviewing the manuscript and for providing positive feedback.
Round 2
Reviewer 1 Report
The author has improved somewhat the manuscript by answering all the reviewers’ questions. Still, I found the answers somewhat shallow and without in-depth analysis. We have already excellent reviews on training immunity that are already published and there is no need to add another superficial account. This review is not novel or insightful and has still misconceptions. Indeed, in rewriting some of the sections, the author introduced some further misconceptions and mistakes (in bold) that are disqualifying (see below a list that is not exhaustive). Evidently, the author is not well versed in immunology of infectious diseases and the molecular biology of epigenetic modifications and should seek co-authors with expertise in the topic.
Disqualifier 1. Adaptive immunity is triggered when the invading pathogen overpowers the nonspecific innate response. This cannot be further from the correct interpretation. Invasion by a pathogen encounters first the natural defenses including the innate immune responses that prepare the adaptive host response, first the primary response and then the secondary response. This is what classical vaccines do. There is always immunological memory after a primary encounter. Nothing needs to be overpowered.
Disqualifier 2. A timely and robust innate immune response can impede the invasion and clearance of respiratory pathogens to attenuate disease symptoms or severity. This indeed is a contradictory statement. Does it impede invasion or clearance? It cannot be both. Actually, overt disease is impeded as some colonization and restricted invasion may occur to trigger the immune system.
Disqualifier 3: Vaccines elicit adaptive lymphocyte-mediated immunity with humoral memory to produce antibodies. Vaccines may elicit both cell-mediated and humoral immunity, in some cases predominantly one or the other. The statement is incorrect because it suggests that the role of lymphocytes is to only deliver antibodies (a Th2 response). They are also very important in the Th1/Th17 and the CD8 response.
Disqualifier 4: It is important to note that these epigenetic alterations in innate immune cells are reversible without altering the DNA sequences. As pointed out by Reviewer 2, epigenetic alterations are not always reversible. In response, the author indicates that this sentence refers to immune-related genetic changes. However, this applies to all epigenetic changes: depending on the situation, reversion may or may not occur. A more detailed explanation is required to address the issue of reversibility.
Author Response
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Author Response File: Author Response.docx
Reviewer 3 Report
Thank you for addressing concerns.
Author Response
I thank the reviewer for your valuable time in providing feedback and consideration to proceed with publication.
