Title: Serum O-glycosylated hepatitis B surface antigen levels in patients with chronic hepatitis
B during nucleos(t)ide analog therapy
Version: 1 Date: 29 Mar 2022
Reviewer's report:
Murata et al described the serum kinetics of a novel assay, the O-glycosylated MHBsAg, in
47 HBV-infected patients at baseline and 48 weeks of antiviral treatment. Correlations with
other viral biomarkers were made. They also attempted to demonstrate the nature of ‘virion’-
identification by this assay. This is an interesting paper and there are increasing discussions
about the detection of various components of HBsAg to predict treatment response and outcome.
I have the following comments:
1/ Introduction: Background: the global prevalence of HBV infection needs to be updated (refer
to either the latest WHO 2022 report or the Polaris Observatory report in 2018).
2/ Methods: What were the treatment indications for the subjects? The baseline characteristics of
these subjects (age, gender, alanine aminotransferase, cirrhosis) should be tabulated, including
separate columns for all patients HBeAg+ and HBeAg- patients. In addition, the baseline levels
of each viral marker should be included in the Table.
3/ Methods: Was serum HBV RNA measured? Also, what were the LLOQ for serum HBsAg,
HBeAg and HBcrAg? I could not find the quantitative HBeAg data (P.7 line 100).
4/ Methods: P.8: line 109-115: I think the authors can describe more clearly what do the
‘samples’ refer to. For readers who are clinicians with minimal laboratory experience may not
find this paragraph a bit hard to follow. For instance, the ‘Serum samples (2uL)’ in line 110
– are these referring to the sample from study subjects? Then, ‘Standard samples (purified M-
HBsAg)’ in line 113 - I believe these are synthetic ones for calibration. Then, ‘The samples (100
uL)’ in line 115 – I think the authors are referring to samples from study subjects again? May
consider more specific description, and rearranging the sentences.
5/ Results: In the supernatant fractions, what were the levels of HBsAg? And was HBV RNA
detectable?
6/ Results: In the immunoprecipitated fractions, the authors mentioned that the HBV RNA level
did not show a significant difference (5.98 vs 6.07 log copies/mL) at 48 weeks of treatment. This
finding is unexpected as HBV RNA is widely reported to be suppressed by NAs, just to a lesser
extent compared to HBV DNA. What would be the reasons behind this observation? Also, why
would HBV DNA still be detectable in the immunoprecipitant despite undetectable serum HBV
DNA? It would be helpful if more sensitive DNA assays for serum measurement were used.
7/ More experiments or analyses are required to convince that the immunoprecipitant consisted
of only DNA/RNA-containing virion+O-glycoslated MHBsAg+ anti-glyco-PreS2. Were more
detailed examination of the immunoprecipitant carried out to confirm the immune complexes
did not carry other source of HBsAg?
8/ Discussion: The authors should explain further elaborate the potential use of this assay
clinically. For example, what groups of patients would benefit from this assay? Contemplating
NA treatment cessation? Prediction of HBsAg seroclearance?
9/ Figure legends (Fig 2, 4 and 5): the light gray circles are referring to HBeAg-negative while
the dark gray circles are referring to HBeAg-positive patients?
Title: Serum O-glycosylated hepatitis B surface antigen levels in patients with chronic hepatitis
B during nucleos(t)ide analog therapy
Version: 1 Date: 29 Mar 2022
Reviewer's report:
Murata et al described the serum kinetics of a novel assay, the O-glycosylated MHBsAg, in
47 HBV-infected patients at baseline and 48 weeks of antiviral treatment. Correlations with
other viral biomarkers were made. They also attempted to demonstrate the nature of ‘virion’-
identification by this assay. This is an interesting paper and there are increasing discussions
about the detection of various components of HBsAg to predict treatment response and outcome.
I have the following comments:
1/ Introduction: Background: the global prevalence of HBV infection needs to be updated (refer
to either the latest WHO 2022 report or the Polaris Observatory report in 2018).
2/ Methods: What were the treatment indications for the subjects? The baseline characteristics of
these subjects (age, gender, alanine aminotransferase, cirrhosis) should be tabulated, including
separate columns for all patients HBeAg+ and HBeAg- patients. In addition, the baseline levels
of each viral marker should be included in the Table.
3/ Methods: Was serum HBV RNA measured? Also, what were the LLOQ for serum HBsAg,
HBeAg and HBcrAg? I could not find the quantitative HBeAg data (P.7 line 100).
4/ Methods: P.8: line 109-115: I think the authors can describe more clearly what do the
‘samples’ refer to. For readers who are clinicians with minimal laboratory experience may not
find this paragraph a bit hard to follow. For instance, the ‘Serum samples (2uL)’ in line 110
– are these referring to the sample from study subjects? Then, ‘Standard samples (purified M-
HBsAg)’ in line 113 - I believe these are synthetic ones for calibration. Then, ‘The samples (100
uL)’ in line 115 – I think the authors are referring to samples from study subjects again? May
consider more specific description, and rearranging the sentences.
5/ Results: In the supernatant fractions, what were the levels of HBsAg? And was HBV RNA
detectable?
6/ Results: In the immunoprecipitated fractions, the authors mentioned that the HBV RNA level
did not show a significant difference (5.98 vs 6.07 log copies/mL) at 48 weeks of treatment. This
finding is unexpected as HBV RNA is widely reported to be suppressed by NAs, just to a lesser
extent compared to HBV DNA. What would be the reasons behind this observation? Also, why
would HBV DNA still be detectable in the immunoprecipitant despite undetectable serum HBV
DNA? It would be helpful if more sensitive DNA assays for serum measurement were used.
7/ More experiments or analyses are required to convince that the immunoprecipitant consisted
of only DNA/RNA-containing virion+O-glycoslated MHBsAg+ anti-glyco-PreS2. Were more
detailed examination of the immunoprecipitant carried out to confirm the immune complexes
did not carry other source of HBsAg?
8/ Discussion: The authors should explain further elaborate the potential use of this assay
clinically. For example, what groups of patients would benefit from this assay? Contemplating
NA treatment cessation? Prediction of HBsAg seroclearance?
9/ Figure legends (Fig 2, 4 and 5): the light gray circles are referring to HBeAg-negative while
the dark gray circles are referring to HBeAg-positive patients?
Please review this paper.
user
Title: Serum O-glycosylated hepatitis B surface antigen levels in patients with chronic hepatitis
B during nucleos(t)ide analog therapy
Version: 1 Date: 29 Mar 2022
Reviewer's report:
Murata et al described the serum kinetics of a novel assay, the O-glycosylated MHBsAg, in
47 HBV-infected patients at baseline and 48 weeks of antiviral treatment. Correlations with
other viral biomarkers were made. They also attempted to demonstrate the nature of ‘virion’-
identification by this assay. This is an interesting paper and there are increasing discussions
about the detection of various components of HBsAg to predict treatment response and outcome.
I have the following comments:
1/ Introduction: Background: the global prevalence of HBV infection needs to be updated (refer
to either the latest WHO 2022 report or the Polaris Observatory report in 2018).
2/ Methods: What were the treatment indications for the subjects? The baseline characteristics of
these subjects (age, gender, alanine aminotransferase, cirrhosis) should be tabulated, including
separate columns for all patients HBeAg+ and HBeAg- patients. In addition, the baseline levels
of each viral marker should be included in the Table.
3/ Methods: Was serum HBV RNA measured? Also, what were the LLOQ for serum HBsAg,
HBeAg and HBcrAg? I could not find the quantitative HBeAg data (P.7 line 100).
4/ Methods: P.8: line 109-115: I think the authors can describe more clearly what do the
‘samples’ refer to. For readers who are clinicians with minimal laboratory experience may not
find this paragraph a bit hard to follow. For instance, the ‘Serum samples (2uL)’ in line 110
– are these referring to the sample from study subjects? Then, ‘Standard samples (purified M-
HBsAg)’ in line 113 - I believe these are synthetic ones for calibration. Then, ‘The samples (100
uL)’ in line 115 – I think the authors are referring to samples from study subjects again? May
consider more specific description, and rearranging the sentences.
5/ Results: In the supernatant fractions, what were the levels of HBsAg? And was HBV RNA
detectable?
6/ Results: In the immunoprecipitated fractions, the authors mentioned that the HBV RNA level
did not show a significant difference (5.98 vs 6.07 log copies/mL) at 48 weeks of treatment. This
finding is unexpected as HBV RNA is widely reported to be suppressed by NAs, just to a lesser
extent compared to HBV DNA. What would be the reasons behind this observation? Also, why
would HBV DNA still be detectable in the immunoprecipitant despite undetectable serum HBV
DNA? It would be helpful if more sensitive DNA assays for serum measurement were used.
7/ More experiments or analyses are required to convince that the immunoprecipitant consisted
of only DNA/RNA-containing virion+O-glycoslated MHBsAg+ anti-glyco-PreS2. Were more
detailed examination of the immunoprecipitant carried out to confirm the immune complexes
did not carry other source of HBsAg?
8/ Discussion: The authors should explain further elaborate the potential use of this assay
clinically. For example, what groups of patients would benefit from this assay? Contemplating
NA treatment cessation? Prediction of HBsAg seroclearance?
9/ Figure legends (Fig 2, 4 and 5): the light gray circles are referring to HBeAg-negative while
the dark gray circles are referring to HBeAg-positive patients?
assistant