PONE-D-23-18377Pangenomic antiviral effect of REP 2139 in CRISPR/Cas9 engineered cell lines expressing hepatitis B virus HBsAgPLOS ONE

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Reviewers' comments:

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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: No

Reviewer #2: Partly

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

Reviewer #1: N/A

Reviewer #2: No

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

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: In this study, Angelo et al investigated the antiviral effect of REP2139 on HBsAg expression/secretion in various engineered HepG2 cell lines expressing HBsAg from different genotypes. Although previous studies have included genotypes A, C, and D, in this study the HBsAg from the most prevalent genotypes (A, B, C, D, E, G), which account for over 96% of human cases, were included. The authors found that REP 2139 exerts antiviral effects on all genotypes, as well as on the vaccine escape mutant. This study is neither comprehensive nor innovative. I have several major comments below to improve this manuscript:

Major comments:

1. The authors only showed the effects in the stable HepG2 cell lines. To further strengthen the conclusion from stable cell lines, the authors need to use infection systems (i.e., HepG2-NTCP and/or primary human hepatocytes) to confirm their finds.

2. In Fig 1, the phylogenetic tree of L-HBsAg is not sufficient. The authors need to include the phylogenetic analyses of HBV polymerase ORF and the full-length DNA sequence of these strains.

3. In Fig 2, it is not surprising that the authors’ construct expressing HBsAg would not support HBsAg secretion. Because there is not polyadenylation signal for HBsAg RNA transcription on this construct. Flipping of HBx sequence alone does not sufficient to conclude that HRPE is important as it also affected the polyA signal sequence. The authors need to dissect the element of polyA signal and HRPE individually from HBx coding sequence in this experiment.

4. REP2139 inhibits HBsAg SVP assembly and secretion, so it should not affect vaccine escape mutant D144A. In addition, the previous study already included some genotypes (A, C, and D). The only additional novel information from this study is genotypes B, E, and G. To improve the novelty of this study, investigations on mutants that affect the SVP assembly and secretion, eg., amino acid between 156 and 169 of S-HBsAg are suggested (Yang et al., 2021, J Virol, doi:10.1128/jvi.02399-20).

Minor point:

1. The sentence in page 3, line 44 “Importantly, HBsAg elimination from blood is required for achievement of functional cure [5] and cessation of therapy [6].” is controversial. It is impossible to eliminate HBsAg from the integrated HBV DNA. It might be better to change it into “Importantly, HBsAg elimination from blood is considered as the hallmark of achieving functional cure [5].”.

2. Page 3 Line 52-53, “In the most recent phase IIA trial of NAP-based combination therapy, durable immunological control of infection in the absence of therapy was achieved in 78% of participants, with 39% of participants further achieving functional cure [10]. How to define the immunological control? By seroconversion or by virological control? In ref 10, it states the detection of HBsAg below the LLOQ. Thus, the authors should use virological control here.

Reviewer #2: Overview:

The authors have shown successful generation of different genotype cell lines (GCLs) expressing hepatitis B surface antigen (HBsAg). This manuscript is the first to demonstrate the pangenomic antiviral effect of a nucleic acid polymer (NAP) such as REP 2139 against HBsAg expression. The results also demonstrated an antiviral effect in a vaccine escape mutant implying that NAPs could have an influence on escape mutants. However, the GCLs that were generated did not incorporate entire HBV genomes and could therefore not recapitulate natural viral replication. Although REP 2139 seems to have an antiviral effect on HBsAg from different HBV genotypes, this effect may be different during natural infection. In its current form, the manuscript is not suitable for publication. Points that need to be addressed are below.

Major points

Title:

1. The manuscript is titled ‘Pangenomic antiviral effect of REP 2139 in CRISPR/Cas9 engineered cell lines expressing hepatitis B virus HBsAg’. The title needs to be rephrased to something along the lines of ‘Pangenomic antiviral effect of REP 2139 in CRISPR/Cas9 engineered cell lines expressing hepatitis B virus surface antigen’. The title indicates that the cell lines replicated HBV, whereas it was only hepatitis B surface antigen (HBsAg) that was expressed.

Abstract and Introduction:

1. The significance of NAPs in clinical trials was mentioned, however the link between NAPs and how they target subviral particles (SVPs) was not described. Previously described mechanisms (found in references 7, 11 and 12) should be included in this manuscript.

2. Authors should describe clearly that the NAP used in the manuscript is REP 2139 and provide more background on this compound.

3. The structure of the abstract and introduction should be revised. Background information on HBV should include the different genotypes followed by the description of NAPs and then conclude with the importance of REP 2139 and the relevance of this NAP in the experiments conducted.

Materials and Methods

1. While the authors have included the relevant methods, several important details have not been described. Seeding and confluency of the GCLs as well as the HepG2 cells were not indicated for CRISPR/Cas9 knock-in, confocal fluorescence microscopy and ELISA experiments. The time points were also not listed for any of the confocal fluorescence microscopy and ELISA experiments. Overall, very little information is provided and these experiments will not be able to be repeated.

2. Statistical analyses were not performed throughout the manuscript and should be described in detail in the Materials and Methods section.

3. Lines 108-117: Plasmids and cloning. The authors need to elaborate further on how the cell lines were generated once pAAVS1-HBsAg-HBx forward were cloned (for the different genotypes)

4. Lines 126-127: Touch down PCR. The authors should specify how normalization was carried out.

5. Lines 136-141: Cell viability. The authors should clarify how the BCA would determine cell viability. This is unconventional and of questionable reliability.

Results

1. Figure 1: Phylogenic classification of L-HBsAg proteins should be placed in the supplementary section. Information provided in the figure is already well established.

2. Figure 2B: What was the significance of including the HBx ORF in the reverse orientation? The HPRE has already been shown to play an important role. The arrows annotating the HPRE orientation are also incorrectly represented. They should be reversed points towards the right for 5’ to 3’ forward orientation.

3. Figure 2C and Figure 4B: The y-axis has an abbreviation ‘Sup HBsAg (RU)’ which has not been fully described in the figure legend. Results from the HepG2.2 15 cells appear to be normalised. An explanation for this and statistical analyses should be included.

4. The authors indicate the insertion of the HBsAg-HBx ORF into the AAVS1 safe-harbour site in Figure 3 and only provide digestion restriction results to ascertain the PCR products. Sequencing should be performed to further validate the presence of genotype-specific sequences and their correct integration.

5. The authors should generate entire HBV-replicating cell lines to assess pangenomic effects more accurately.

6. Figure legends for Figures 3, 4 and 5 are not comprehensive enough.

7. BCA assay was stated to be performed in the methodology section (Line 136 to 141) to assess cell viability however data for this assay has not been provided.

8. Line 188: Provide reference.

9. Lines 236-237: ‘Importantly, we observed that the inhibition of HBsAg secretion after REP 2139 treatment is faster and greater in the HepG2.2.15 cell than in all GCLs’. Although greater inhibition of HBsAg secretion after REP 2139 treatment was observed in HepG2.2.15 cells (Figure 5), it is misleading to state that ‘faster’ inhibition was observed.

Discussion

1. Lines 266-267: This section covers important concepts and should be explained more thoroughly.

2. Lines 277-281: This explanation is not clear and should be rephrased.

Conclusion

1. Lines 296-297: Time course data has not been shown to verify stable expression of HBsAg from the GCLs.

Minor Points

1. Line 20: Mention SVP as non-infectious and Dane as infectious.

2. Line 21: Write out HBsAg in full, change “admitted” to “considered”.

3. Line 30: REP 2139 is not referred to as a NAP.

4. Line 31: Remove the word “in” “in the vaccine escape mutant”.

5. Line 39: Remove 2019.

6. Line 46: Discuss the structure of the NAPs in greater detail and link it to the SVPs.

7. Line 56: Rewrite “in a subset of genotypes including genotype D [8, 10-12], and genotypes C and A [8]” to “in a subset of genotypes including genotypes A, C and D [8-12].

8. Line 64: Remove “in different geographical regions.”

9. Line 73: Provide reference.

10. Line 87: As previously mentioned, provide context for REP 2139.

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Reviewer #1: No

Reviewer #2: No

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Pangenomic antiviral effect of REP 2139 in CRISPR/Cas9 engineered cell lines expressing hepatitis B virus surface antigen.

PONE-D-23-18377R1

Dear Dr. Labonté,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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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: (No Response)

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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: Yes

Reviewer #2: No

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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 appropriately addressed the concerns. Although with low novelty of this study, the story is complete and suitable for publication in PLoS One.

Reviewer #2: The manuscript is improved as a result of the revisions. Minor changes to finalise are the following:

1. LA description of how NAPs function is still missing. If mechanisms are not fully understood, this should be stated.

2. Some Supp. Figures do not seem to be cited in the body of the MS.

3. An explanation for why S Ag expression is adequate instead of complete replication of the virus should be included in the text.

4. Stats are missing from Fig. 4.

5. The conclusion states that stable expression for more than 4 months was achieved. However, the MS does not provide the relevant data. (This sentence starting on line 338 also needs editing.)

6. Methodology used to generate the data described in Fig. 5 seems to be missing.

Reverse orientation of sequences depicted in Fig. 2B is unconventional.

Line 33 ‘into’ needs to be replaced with ‘in’

Line 66-67 should be added to the previous paragraph.

Line 116, ‘REP 2139 antiviral effect is’ change to REP 2139 antiviral effect was’

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Reviewer #1: No

Reviewer #2: No

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