Adenovirus—Extracellular Protein Interactions and Their Impact on Innate Immune Responses by Human Mononuclear Phagocytes
Round 1
Reviewer 1 Report
This is a valuable, well updated review on an interesting subject.
Comments: line37 give reference to very high doses. line 40 oral
line 63 AdV 6 is not a common type.
,line 65 HAdV-IC were not detected after death in children " that have succumb to HAdV" but rather during infection in children with severe disease line 69 give reference to. us and or others;
Line 81 which cells does which refer to.line117 induced;line 141 where ,on which capsid proteins are the negative areas located that Au refers to? Toogood and Hay once described distinct differences between hexons from Ad5 and Ad40;
line 126 it is unknown (if in man)?
lines 165 166 as Au know both HAdv 5 and 26 are covid 19 vaccine candidates can the difference noted in mice also be confirmed in man?
line 170 does this effect require dodecons?
line 178 HAdV E4 is not a simian virus It is a human virus originally the only member of species E. This species has recently accomodated several newly described chimpanzee adenoviruses.
line 179 180 Did Yerold Gordon show that LL-37did not inhibit the propagation of HAdV B3, C5 and D8 or did he not study them?
line 197 which valuable data has A Baker provided?
line 216 infected
Are any of the described interactions studied on transcriptome levels?
L
Author Response
Reviewer 1
Line 37 give reference to very high doses.
The text has been modified to include this information.
Line 40 oral
The text has been modified.
Line 65 HAdV-IC were not detected after death in children "that have succumb to HAdV" but rather during
infection in children with severe disease.
The text has been modified.
Line 69 give reference to “us and or others”.
References added
Line 81 which cells does which refer to.
The text has been modified.
Line 126 it is unknown (if in man)?
We cannot find information addressing the case in humans.
Line 141 where? on which capsid proteins are the negative areas located that Au refers to? Toogood and
Hay once described distinct differences between hexons from Ad5 and Ad40.
The text has been modified to include this information.
Lines 165 166 as Au know both HAdv 5 and 26 are covid 19 vaccine candidates: can the difference noted in
mice also be confirmed in man?
We have a m/s in preparation that shows a differential effect between Lf and defensins on Ad5, 26 and 35 in human
phagocytes. Unfortunately, this this review is not the place to expand on these points.
Line 170 does this effect require dodecons?
Thank you for the comment. The text has been modified to include this information.
Line 178 HAdV E4 is not a simian virus It is a human virus originally the only member of species E. This
species has recently accommodated several newly described chimpanzee adenoviruses.
The text has been modified.
Line 179 180 Did Y Gordon show that LL-37did not inhibit the propagation of HAdV B3, C5 and D8 or did he
not study them?
HAdV B3, C5 and D8 were included in the study but did not show a statistical difference
Line 197 which valuable data has A Baker provided?
The text has been modified to include this information.
Are any of the described interactions studied on transcriptome levels?
Not that we can find.
Reviewer 2 Report
An excellent and comprehensive review. My recommendation is “accept with very minor revisions”.
The manuscript by Cheneaus C and Kremer EJ is a very well written, easy-to-read, and comprehensive overview of the human adenovirus interactions with extracellular proteins and its impact on the innate immune response. I found the manuscript highly appropriate and relevant for this special issue of Viruses and believe, it will benefit both adenovirologists and broad readership.
Although the manuscript is well written, I still have a few minor comments, which the authors may take into consideration:
Manuscript format related suggestions:
Line 2, 3, and 4: Title, the first letter of every word should be in the capital. This applies to headings as well for example Antimicrobial Peptides.
Line 5: and not &
Line 6 and 7: Each author's email address should be given here. Also, check the affiliation format.
Reference numbering format. AB1,2 or AB [1,2].
Missing: Author Contributions, Funding, Acknowledgements, and Conflicts of Interest
Suggestions related to content:
General: Could the authors provide an illustration, summerizing the interaction between HAdVs and extracellular proteins? Just a suggestion.
Line 20: HAdVs
Line 19-34: More references required. Although the first paragraph provides a general overview of adenoviruses and the host immune response, I would still suggest the authors provide more references. For example, the following statement needs reference: In most cases, human AdVs (HAdVs) cause self-limiting respiratory, ocular, or gastrointestinal tract infections in all populations regardless of health standards.
Line 24: Adenoviruses (AdVs)
Line 26: HAdVs already defined, no need to define again (human AdVs)
Line 31-34: The following statement can mislead the readers, as SARS-CoV-2 and malaria vaccines are based on Chimpanzee AdVs. HAdV-based vaccines, including live virus or replication-defective vectors, are used to prevent acute respiratory disease in military recruits5 (e.g. HAdV type 4 (HAdV-E4) and HAdV-B7) or in trials targeting Malaria, Ebola virus, and SARS-CoV-2.
Line 44: incorporated not incorporate
Line 46-54: References required.
Line 110: Complement or Complement System
Line 131: Used antimicrobial. Antibiotic represent only anti-bacterial molecules.
Line 168-169: Reformate the sentence.
Line 171: types 1, 2, 5, and 6
Line 176: The authors stated that “…..but has no effect on HAdV-C2 infection of these cells (75), however the finding of ref. 75 is opposite to what the authors have stated. The original article states that “We show that the antiviral protein bovine lactoferrin enhances infection of monocyte-derived dendritic cells (MDDC) by adenovirus species C serotype 2 (2C) isolates”. Clarify concerning bovine and human lactoferrin.
Line 177-180: Can the authors provide any potential explanation (one sentence or two) on these contradictory outcomes? Have researchers from these two studies (ref. 76 and 77) used different cell-lines, strains, etc.
Line 187: The authors stated “Numerous studies” but then cite only one study.
Line: 202: HAdVs?
Line 223: phagocytes? Or phagocytes or will…..
Line 228-229: More references are required or authors may site review articles.
Author Response
Reviewer 2
Line 2, 3, and 4: Title, the first letter of every word should be in the capital.
Changed
This applies to headings as well for example Antimicrobial Peptides.
Changed
Line 6 and 7: Each author's email address should be given here. Also, check the affiliation format. Reference
numbering format. AB1,2 or AB [1,2].
Missing: Author Contributions, Funding, Acknowledgements, and Conflicts of Interest
Changed
Line 19-34: More references required. Although the first paragraph provides a general overview of
adenoviruses and the host immune response, I would still suggest the authors provide more references. For
example, the following statement needs reference: In most cases, human AdVs (HAdVs) cause self-limiting
respiratory, ocular, or gastrointestinal tract infections in all populations regardless of health standards.
More references were added and the text changed
Line 31-34: The following statement can mislead the readers, as SARS-CoV-2CoV-2 and malaria vaccines are
based on Chimpanzee AdVs.
This comment is incorrect: SARS-CoV-2 vaccines are Ad26-based (J&J & Russian) or Ad5-based (Russian &
Chinese) and ChAd-based (Oxford). Malaria vaccines are also Ad35-based.
References Added
Line 46-54: References required.
References added
Line 168-169: Reformate the sentence.
Reformatted
Line 176: The authors stated that “….. but has no effect on HAdV-C2 infection of these cells (75), however the
finding of ref. 75 is opposite to what the authors have stated. The original article states that “We show that
the antiviral protein bovine lactoferrin enhances infection of monocyte-derived dendritic cells (MDDC) by
adenovirus species C serotype 2 (2C) isolates”. Clarify concerning bovine and human lactoferrin.
The text has been revised.
Line 177-180: Can the authors provide any potential explanation (one sentence or two) on these contradictory
outcomes? Have researchers from these two studies (ref. 76 and 77) used different cell-lines, strains, etc.
A difference was in the order of infection
Ref 108: they add HAdV to A549 cells, washed, and then added LL-37.
HAdV (to obtain a virus input of 5 PFU per well) was added to each well. After 2 h at 37°C, the virus suspension was
aspirated, and the medium was replaced with serial dilutions of the test compounds -- concentration of LL-37 was
determined between 1/100 and 1/50 CC50 of LL-37, 0, 55, 110, 220, and 440 μg/mL.
Ref 109: they make and add AdV+LL-37 complexes to A549 cells.
(In a single tube, each virus sample (∼104 pfu) was incubated directly with LL-37 (500 μg/ml, 111 μM)
Read out: time-kill direct inactivation assay.
Line 187: The authors stated “Numerous studies” but then cite only one study.
References added
Line 223: phagocytes? Or phagocytes or will…..
Sentence modified
Line 228-229: More references are required or authors may site review articles.
References added
Reviewer 3 Report
In this review, the authors provide a synopsis of the soluble protein components of the innate immunity involved in interactions during HAdV infection with phagocytic cells. The contents of this MS are overall informative and a valuable contribution on a so-far lesser reported aspect of anti-adenoviral responses, which makes it suitable for publication.
My remarks are therefore mostly directed towards a few stylistic improvements, as well as revisions on some inaccurate information given in the manuscript, which I feel should be addressed before final consideration.
More attention and input are still needed with regard to some virological aspects, especially in the following passages:
Line 24: Please revise the information on genome length. Fish (Ichtadenovirus): currently longest known genome with over 48kB; on the other hand in the genus Siadenovirus genomes have by now been determined with less than 26kB.
Line 26: change “isolated” to “obtained/recovered from”. Many of the adenovirus findings, especially those from non-human hosts, are not per se isolated (in vitro)
Line 27: “…in all populations regardless of health standards”: please explain, or define more accurately, what this means? A self-limiting character of HAdV infections is based on the immune status of the individual patient, so the terms “populations” and “health standards” seem a bit misplaced in this context.
Line 28: for species designation I would advise to follow the official nomenclature guidelines, at least at the first mentioning (Human adenovirus A to Human adenovirus G, HAdV-A to HAdV-G)
Line 28: The information on the existence of 88 types is substantially outdated; with new sequence contributions adding up rapidly, there should be well over 100 types of HAdV by now. Please verify.
Line 177: perhaps double-check that the terms “propagation” and “infection” are not used synonymously, virologically these are two distinct mechanisms
Many passages contain imprecise phrasiology, are slightly unstructured or might be misleading.
Of course this is a matter of personal preference, but more colloquial expressions should be avoided and replaced with a more scientific and/or formal terminology. For instance:
Line 200: “The literature is muddled with citations that mix rodent, nonhuman primate and human data” – what is meant by “rodent data” or “human data”? This is a very vague formulation; perhaps also avoid the word “muddled”.
Line 214: “location” – site (or route) of infection/administration
Also not sure if “time” (lines 214, 219) expresses adequately what the authors mean to say. Maybe chronological order/appearance of extracellular proteins? Or timecourse/timeline of extracellular proteins secreted?
Line 10: I recommend to specify at first mentioning: “soluble extracellular proteins associated with the innate immune response”
Line 20: A handful of: replace by “an array of”, or similar
Line 42: “In these varied environments, the immune cells present at the time of injection, or recruited to the site of delivery, are not the same and so, the responses differ.”: change to: “Variations in frequency and activity of immune cell populations present, or migrating, to different sites of delivery lead to differences in responses” (or similar)
Line 111, the three pathways of complement activation are mentioned, but it would be helpful to specify those pathways which are of significance in the response against Ads. The lectin pathway is more relevant for bacterial pathogen recognition.
The complete section 4 (AMPs) is relatively difficult to follow due to the frequent switches between the different categories of AMPs and their functions. Maybe this could be structured more coherently.
Line 191: “The use of coagulation factors by HAdV may be of importance not only for the liver tropism but also during primary infections by wild type viruses”: it is not clear what virus´ liver tropism, as opposed to wt viruses; I assume from the context of the aforementioned study that authors mean the liver tropism of the Ad-5 vector, but it is not mentioned explicitly.
Line 207: “human FX and HAdVs”: change to “human FX-HAdV complexes”
Line 14: avoid the “±” in the text (in the presence or absence of…)
In the introduction, in context of Ad applications, the authors might consider to add some background information on rAds. Since information is provided on live virus vaccines, another passage could also be added on Ad vectors (e.g. the main reasons justifying the interest in adenoviruses for vectorization, the current distribution of adenoviral vectors, which types are commonly used etc.). Perhaps some useful information can be obtained from a review by Lee at al., 2017 (Genes Dis.) or other relevant works.
A few further, minor suggestions and corrections:
Lines 9, 20: human adenovirus (HAdV). I suggest to use the singular instead of the plural form, referring to the virus in a general context
human adenovirus (HAdV) or HAdV-based vectors (change “and” to “or”)
Line 25: impact on human health
Line 28: change “classed” to “determined/identified/distinguished”
Line 35: data…demonstrate
Line 39: mucosa (orotracheal tract, gastrointestinal or ocular tracts)
Line 44: needs to be incorporated
Also check the verb flexion in other cases, I spotted this repeatedly (e.g. lines 216, 219)
Line 59: the abbreviation “Ab” should be specified at first mentioning
Line 154: exhibits immunomodulatory effects
Line 157: by its chemotactic activity
Line 164: lung epithelium/lung epithelial cells
Line 186: …to some of the hexon hypervariable regions
Line 217: “…may be recruited only to specific organs…”
Line 232: what happens
Line 233: should it be: in what ratio (?)
As for the discussion, I would be interested, to which extent findings based on adenoviral vectors can be compared to wild type virus infections? Many of the data discussed in this review derive from studies with vectors, and the authors have repeatedly pointed out that such findings may not be directly applicable to live virus infections. Are there, for instance, models which have studied extracellular proteins and their effects on immunogenicity and tropism in wt-HAdV infections (or live vaccines), with similar results available for the analogous vector (administered via a different route than natural infection would occur through)?
Author Response
Reviewer 3
Line 24: Please revise the information on genome length. Fish (Ichtadenovirus): currently longest known genome with over 48kB; on the other hand in the genus Siadenovirus genomes have by now been determined with less than 26kB.
The text has been modified.
Line 28: The information on the existence of 88 types is substantially outdated; with new sequence contributions adding up rapidly, there should be well over 100 types of HAdV by now. Please verify.
cc: more than 100 types
The text has been modified.
Line 28: for species designation I would advise to follow the official nomenclature guidelines, at least at the first mentioning (Human adenovirus A to Human adenovirus G, HAdV-A to HAdV-G).
The text has been modified.
Lines 9, 20: human adenovirus (HAdV). I suggest to use the singular instead of the plural form, referring to
the virus in a general context.
The text has been modified.
Line 10: I recommend to specify at first mentioning: “soluble extracellular proteins associated with the innate
immune response”.
The text has been modified.
Line 14: avoid the “±” in the text (in the presence or absence of…).
The text has been modified.
Line 27: “…in all populations regardless of health standards”: please explain, or define more accurately, what
this means? A self-limiting character of HAdV infections is based on the immune status of the individual
patient, so the terms “populations” and “health standards” seem a bit misplaced in this context.
The text has been modified.
Line 42: “In these varied environments, the immune cells present at the time of injection, or recruited to the
site of delivery, are not the same and so, the responses differ.”: change to: “Variations in frequency and
activity of immune cell populations present, or migrating, to different sites of delivery lead to differences in
responses” (or similar).
The text has been modified.
Line 111, the three pathways of complement activation are mentioned, but it would be helpful to specify those
pathways which are of significance in the response against Ads. The lectin pathway is more relevant for
bacterial pathogen recognition. The complete section 4 (AMPs) is relatively difficult to follow due to the
frequent switches between the different categories of AMPs and their functions. Maybe this could be
structured more coherently.
The complement section has been rewritten
Line 177: perhaps double-check that the terms “propagation” and “infection” are not used synonymously,
virologically these are two distinct mechanisms.
We have double-checked.
Line 191: “The use of coagulation factors by HAdV may be of importance not only for the liver tropism but
also during primary infections by wild type viruses”: it is not clear what virus´ liver tropism, as opposed to wt
viruses; I assume from the context of the aforementioned study that authors mean the liver tropism of the Ad-
5 vector, but it is not mentioned explicitly.
The text has been modified.
Line 200: “The literature is muddled with citations that mix rodent, nonhuman primate and human data” –
what is meant by “rodent data” or “human data”? This is a very vague formulation; perhaps also avoid the
word “muddled”.
The text has been modified.
In the introduction, in context of Ad applications, the authors might consider to add some background
information on rAds. Since information is provided on live virus vaccines, another passage could also be
added on Ad vectors (e.g. the main reasons justifying the interest in adenoviruses for vectorization, the
current distribution of adenoviral vectors, which types are commonly used etc.). Perhaps some useful
information can be obtained from a review by Lee at al., 2017 (Genes Dis.) or other relevant works.
We prefer to keep the review focused on the soluble protein-HAdV-phagocyte axis. Adding recombinant HAd vectors
is beyond the scope of this review and also outside our area of expertise
As for the discussion, I would be interested, to which extent findings based on adenoviral vectors can be
compared to wild type virus infections? Many of the data discussed in this review derive from studies with
vectors, and the authors have repeatedly pointed out that such findings may not be directly applicable to live
virus infections.
True, many of the studies used AdV vectors and therefore they cannot exclude the possibility that extracellular
proteins impact propagation. The perspective has been modified to include this caveat.
Are there, for instance, models which have studied extracellular proteins and their effects on immunogenicity
and tropism in wt-HAdV infections (or live vaccines), with similar results available for the analogous vector
(administered via a different route than natural infection would occur through)?
We are unaware of clinical (human) data addressing this interesting issue. However, Smith and colleagues addressed
mouse adenovirus infections and the impact of alpha-defensins. This example has been included in the perspectives.
Reviewer 4 Report
Although not necessary, might be helpful to add a summary table or schematic highlighting which extracellular proteins interact with the immune cell and epithelial cells and downstream response or consequence.
Author Response
Reviewer 4
Schematic highlighting
Great idea but considering the few time we had we decided to focus on the text (and not to do a schematic
highlighting).
