PONE-D-22-20362Characterization of hepatitis B viral forms from patient plasma using velocity gradient: evidence for an excess of capsids in Dane particles enriched fractionsPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The aim of this work is to identify the different types of HBV viral particles circulating in the serum of patients. This simple work is in fact very complicated because all the circulating particles can be relatively close in terms of size and composition. The purification of these different particles is here carried out with several approaches. It should be noted here that two plasmas are HBeAg positive and the other two are HBeAg negative. It is difficult to understand if a significant difference exists between these two types of plasma in the production of all types of viral particles.

The purification system has limitations that the authors could comment. For example, fraction 5 -fig 1- corresponding to dane particles -infectious virus- actually contains many other types of particles as shown in other figures. This sometimes makes the text difficult to understand. Similarly, some paragraphs could be partially rewritten avoiding mixing the results obtained on the two types of plasma (lines 157-173). The low abundance of RNA-containing particles could be discussed in light of the work of J Hu et al showing that empty and infectious particles are enveloped while those containing RNA are not. It is also difficult to say whether these RNA particles are infectious.

Despite these limitations, this work deserves to be published because results reinforce that the plasma of infected people is rich diverse particles. Whether all these particles have a pathophysiological role remains to be elucidated. This type of result has been obtained previously by other groups but with very different approaches, in particular the use of native agarose gel which also has difficulty separating the various types of particles.

Minor Corrections.

First of all it seems important to replace the symbols by colors. It is very difficult to follow the signal of each antigen, especially when the two curves overlap.

Line 126, ‘’’ as percentage for’’’ as percentage of what? Could you clarify?

Page 15, lines 130-132: ‘’’Noteworthy, the richest DNA fraction for each plasma was associated with slightly detectable HBsAg, with values below 17 IU/mL.’’’’ This sentence is unclear. HBsAg is represented by empty squares but its signal at fractions 4-6 seems so weak that we could deduce that VPs do not have HBsAg.

Page 15, lines 139-141 : ‘’’’Surprisingly, on HBeAg negative samples (B7207 and B7686), HBcrAg was also detected and quantified in the last upper two fractions (F13-14) with concentrations around 4.4 log U/ml in each of these fractions’’’’’. I don't understand. In Figure 1D, fractions 11-14, HBcrAg is absent.

And then ‘’ HBeAg negative plasmas were non-reactive for HBeAg when tested with the HBeAg specific assay’’’, If this is not HBeAg, what would it be???

Page 17, Line 166, “””For HBe negative plasmas, these ratios (VPHBS/VPDNA) were reproducibly well above 1 (89 and 1777) despite similar gradient profiles.”””” Does this mean that these particles are particularly rich in protein S?

Page 17, lines 164-173, this whole paragraph is very difficult to understand. Knowing that we started only from the fractions containing the largest amount of DNA ‘’’ the expected number of

VP in the richest DNA fraction for each plasma (4 or 5 depending on the gradient) was calculated according to each measured marker’’’’ this illustrates that the purification conditions are not optimal to separate particles many types of particles being eluted at the same time.

Could you elaborate why NP40 was used in this assay.

Lines 195-202, ‘’’Profiles obtained through equilibrium gradient looked similar to the ones obtained using velocity’’’ If the equilibrium gradient shows similar results as to the equilibrium sucrose density gradient why do you present these data?

Lines 228-230 ’’’This specific profile tends to indicate that this unique HBsAg peak relates to the residual presence of SVP despite the previous velocity separation step’’’. Can this result also explain why in table 2 the VPHBs/VPDNA ratio is 1770??

In the discussion, the presence for which HBeAg (plasma HBeAg+) is located in the upper fraction which actually corresponds to the HBcrAg antigen in the HBeAg- plasma is not clear at all.

Budding capsids is an interesting concept. I do not understand in your work whether these particles are abundant or not (("abundant type" line 377)). In any case, the mechanism of production of these particles will have to be elucidated. Naked particles are produced differently than Dane capsids but they are not enveloped by a lipid bilayer while yours are.

Reviewer #2: The authors analyzed 4 plasma samples (genotype D or E, HBeAg+ or HBeAg-) by nycodenz or sucrose gradient centrifugation, or sequential centrifugation for better separation, with or with NP40 treatment to convert virions into capsids. The purified fractions were analyzed for HBV DNA, HBV RNA, HBcrAg, and HBsAg. Based on their measurements and calculation, they suggested presence of enveloped core particles (virions) lacking HBV DNA or RNA (empty virions; previously reported by Jianming Hu’s lab), enveloped core particles containing HBV RNA (reported before by many groups), in addition to enveloped core particles lacking envelope protein (“budding capsids”; never described before). They also found HBcrAg in fractions lighter than subviral particles even for HBeAg-negative samples, which they attributed to immune complex between HBeAg and anti-HBe. They also calculated that for HBeAg-positive samples, HBeAg accounts for 2/3rds of HBcrAg. In general, this was an interesting study. Some questions:

1. Were all the four plasma samples well preserved? No issue of poor storage and sample degradation?

2. I am not sure if the calculations and conversion of the amount of DNA or protein into capsids, virions, subviral particles are correct (Does one virion contain 180 or 240 copies of core protein? Does it contain 1 or 2 genomes?).

3. The huge excess of subviral particles relative to virions, which become even more serious for the HBeAg-negative samples, may cause contamination by subviral particles in virion-enriched fractions. The latter point was confirmed in Fig. 5, by further centrifugation of a virion-enriched fraction from nycodenz gradient through sucrose cushion. That point is also suggested by the much higher VPHBs/VPDNA ratio and much lower VPHBc/VPHBs ratio from the two HBeAg-negative samples (especially the genotype D sample; B7686) than the two HBeAg-positive samples. Thus, I would tend to more believe in data generated from the HBeAg-positive samples.

4. As the authors discussed, the presence of “budding capsids” requires validation from electron microscopy. Alternatively, zycodenz gradient followed by sucrose gradient centrifugation can be further followed by native agarose gel electrophoresis (NAGE), with subsequent detection of HBcrAg, HBsAg, and HBV DNA. That point can be raised in the Discussion section.

Minor issues: English writing and clarity of presentation need improvement. For example, “in Dane particles enriched fractions” in the title should be “in fractions enriched in Dane particles”. Lines 49-50: “hepadnaviridae family……..gathers DNA reverse transcribing hepatotropic viruses”. “gather” should be changed.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: gradient centrifugation of plasm samples.docx

Characterization of hepatitis B viral forms from patient plasma using velocity gradient: evidence for an excess of capsids in fractions enriched in Dane particles

PONE-D-22-20362R1

Dear Dr. Thibault,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Fabrizio Mammano

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have answered all the questions asked. This resulted in some modifications to the figures and text making this work interesting and worth publishing.

Reviewer #2: The authors have responded to critiques raised in the previous round of review. I have no further comments.

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Reviewer #1: No

Reviewer #2: No

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