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. 2020 Aug 26;15(8):e0237398. doi: 10.1371/journal.pone.0237398

Response to hepatitis B virus vaccination in individuals with chronic hepatitis C virus infection

Ashraf A Ashhab 1, Holly Rodin 2, Marilia Campos 3, Ahmad Abu-Sulb 4, Jane A Hall 2, Jesse Powell 3, Jose D Debes 3,5,*
Editor: Jason Blackard6
PMCID: PMC7449383  PMID: 32845914

Abstract

Background

Previous reports show conflicting results regarding hepatitis B virus (HBV) vaccine efficacy in Hepatitis C virus (HCV)-infected individuals.

Aims

To evaluate HBV-vaccine response and identify possible factors that may contribute to lower vaccine efficacy in patients infected with HCV.

Methods

We retrospectively evaluated all patients with chronic HCV infection at Hennepin County Medical Center, in Minneapolis, Minnesota, between 2002 and 2018. We addressed laboratory, liver-related, virus-related as well as vaccine-related variables, and their association to HBV vaccine response. Differences were tested using either a Chi-squared test or a T test to compare means between the two populations. Multivariate regression was modeled as a logistic regression.

Results

1506 patients were evaluated, of which 525 received appropriate HBV vaccination and were assessed for response. Among those, 79% were vaccine responders and 21% were non-responders. On multivariate analysis, cirrhosis was associated with lower response to the vaccine (OR 0.6, CI 0.44–0.94). We found no significant differences for vaccine response in relation to smoking (87% vs 86%), IV drug abuse (74% vs 72%), Diabetes Mellitus (26% vs 22%) being on hemodialysis (2% vs.5%), or virus related variables.

Conclusion

HCV infection seems to impair HBV vaccine response, with cirrhosis being the only identifiable risk factor for hypo-responsiveness among studied clinical and virus-related variables.

Introduction

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver disease leading to cirrhosis and hepatocellular carcinoma worldwide. The world health organization estimates that in 2015, 257 million people were living with chronic HBV infection, and 71 million were living with chronic HCV [1,2].

Dual infection with HBV and HCV is not uncommon, as both viruses share some common paths of transmission and risk factors, including IV drug use, hemodialysis, and frequent blood transfusions such as in hemophilia [3].

Indeed, more than 25% of HCV-positive patients in the United States had positive markers for hepatitis B exposure, a proportion nearly six times that in the HCV-negative group [4]. Higher rates of cirrhosis and increased severity of liver disease have been reported with co-infection compared to either HBV or HCV mono-infection [5,6]. Additionally, HBV re-activation has been reported in HCV and HBV co-infected individuals receiving direct-acting antiviral (DAA) treatment, including individuals with either current or previous HBV infection [7,8]. Despite the availability of new DAA treatment for HCV and the realistic potential of HCV elimination, many HCV-treated patients continue to exhibit high risk behavior after treatment, putting them at risk for re-infection [9].

As such, HBV vaccine is recommended as the primary means to prevent HBV super-infection and its associated increase in morbidity and mortality in HCV-infected subjects [10].

In this regard, several reports suggest a similar HBV vaccine response rate among HCV infected individuals [11,12], while others suggest that this population mounts a poorer vaccine response [13,14]. This hypo-responsiveness is thought to be related to alterations in the cellular and humoral arms of the immune response secondary to HCV infection [15,16]. The impact of viral related variables such as viral load and viral genotype are controversial [13,17].

In light of the current availability of new DAAs which have effectively turned HCV infection into a curable disease, with well over 90% sustained virological response in most genotypes, attention should be focused towards HCV-related complications and indirect effects, such as poor vaccination responsiveness [18]. In this study we investigated the effect of HCV infection on HBV vaccination response, and the impact of clinical variables, life style, and virus related variables on vaccination response.

Methods

Study design and patients

This is a retrospective observational cohort study conducted through chart review of patients. Characteristics, clinical and virus-related variables of individuals infected with HCV who received HBV vaccination in Hennepin Healthcare, a county hospital in Midwestern US, between the years of 2002–2018 were reviewed.

For this study, 1506 electronic medical records (EMR) of individuals diagnosed with HCV (all available) were reviewed. The institutional review board of Hennepin healthcare approved the study. The data were analyzed anonymously therefore consent was not obtained from individual patients. The primary objective was to assess the response rate of patients with HCV to HBV vaccination, and the impact of clinical and virus related variables on response rate.

Clinical information of the patients were collected from EMR by the participating investigators. The data extracted included patients’ age at the time of Hepatitis B Surface Antibody (HBsAb) test, number of HBV vaccine doses received, potential risk factors (ie, smoking, IV drug use and alcohol use), presence of cirrhosis, Diabetes mellitus (DM), End Stage Renal Disease (ESRD) requiring dialysis, HCV viral load, and HCV genotypes. Patients with a positive hepatitis B core antibody were excluded from the study.

Viral definitions

Patients were considered to have chronic HCV infection if they had a positive polymerase chain reaction assay (PCR) for HCV RNA at the time of receipt of HBV vaccination, and at the time of the HBsAb test check.

A positive HBsAb test was defined as having a titer of 12 mIU/mL or above. Patients were considered to be vaccine responsive if they developed a positive HBsAb test following at least one dose of HBV vaccine. Vaccine non-responders were defined as patients who did not develop a HBsAb following at least 3 vaccine doses. Complete vaccination was performed by injection of 20 μg recombinant HBsAg into the deltoid muscle at months 0, 1 and 6. Patients involved in the study received any of the following brands for HBV vaccination; Glaxo smith kline, Glaxo wellcome, Merck, Smith Beecham, Sanofi, Aventis, Abbot, and JHP. The majority of patients with known dates of the first vaccination dose received the first dose prior to HCV diagnosis (69%), however, most of the patients included in the study expressed a remote history of risk factors for HCV infection, suggesting early acquisition of HCV infection. Patients positive for Hepatitis B core antibody were removed from the analysis.

Clinical definitions

Patients were considered to be cirrhotic at the time of the diagnosis if: a) they had a persistent International normalized ratio (INR) level of > 1.2, persistent high levels of total bilirubin (> 1.2 mg\dL) in addition to low platelets (< 150,000\mm) and radiographic cirrhotic morphology of the liver; b) cirrhotic liver morphology on imaging in addition to an INR of > 1.2, c) A combination of low platelet levels with high total bilirubin levels and an INR of > 1.2 at any time, d) liver biopsy or fibroscan indicating cirrhosis, or e) they had clinical stigmata of end stage liver disease (ESLD) [19]. Patients were considered to have ESRD if they had a glomerular filtration rate (GFR) of less than 15 mL/min/1.73 m2 and were receiving hemodialysis [20]. Patients were placed into a responsive group and a non-responsive group.

Statistics

Statistical analyses were performed using SAS version 9.4. Differences between continuous variables were evaluated at the means using a T-Test. Categorical variables were evaluated using a Chi-Square test. The multivariate regression was modeled as a logistic regression.

Results

Demographics

We reviewed information of 1506 patients with chronic HCV infection. Of those, 85% percent (n = 1276) received HBV vaccination, 50% (n = 646) of which were checked with HBsAb test afterwards.

When comparing the patients who underwent HBsAb testing post vaccination to those who did not, both groups showed male predominance (70% vs 63%, respectively) and the group with post vaccination testing was slightly older, with mean ages of 54 (IQR 53–54) and 48 (IQR 47–50), p = 0.0001, in each groups respectively. Additionally, those who were not tested for HBsAb had lower rates of cirrhosis (14% vs 25%, p = 0.01).

Out of the 646 patients who were evaluated with HBsAb testing, 121 patients were excluded from the analysis as they were non-responders but had received less than 3 HBV vaccine doses.

Among the remaining 525 patients, 80% (n = 418) developed HBsAb after vaccination with any number of HBV vaccine doses (vaccine responders) whereas 20% (n = 107) did not develop the antibody, following 3 or more HBV vaccine doses (vaccine non-responders) (Fig 1). Out of all the patients who received 3 or more doses in both groups, only 50% developed HBsAb.

Fig 1. Vaccine responders and non-responders involved in the final analysis.

Fig 1

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When characterizing patients by the number of the HBV vaccines administered, 23% received 1 vaccine dose, 25% received 2 doses, 37% received 3, and 15% received over 3 HBV vaccines. Among the patients who received 2 doses, the response rate was 17%, compared to 42% in patients that received 3 doses (Table 1). The average number of vaccine doses in responders was 2.6, compared to 3.6 in non-responders (p<0.001) (Table 2).

Table 1. HCV patients’ response following various number of HBV vaccine doses.

Number of HBV vaccine doses Proportion of HCV patients which were vaccinated (n = 413) Proportion of responders (n = 185) Proportion of non-responders to any number of vaccines (n = 228)
1 dose 23% (n = 95) 25% (n = 46) 21% (n = 49)
2 doses 25% (n = 103) 17% (n = 31) 32% (n = 72)
3 doses 37% (n = 153) 42% (n = 79) 32% (n = 74)
≥4 doses 15% (n = 62) 16% (n = 29) 15% (n = 33)
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Table 2. Demographics, clinical and virus related-variables for responders with any number of doses vs non-responders.

Responders (n = 418) Non-responders n = 107 P value
Average age at the time of HBsAB test 52 51 0.6
Males (%) 69% 65% 0.4
Females (%) 31% 36% 0.4
Average number of doses 2.6 3.6 <0.001
Presence of cirrhosis (%) 24% 29% 0.4
Alcohol use (%) 79% 75% 0.4
Tobacco use (%) 87% 86% 0.8
IV drug use (%) 74% 72% 0.6
Diabetes (%) 26% 22% 0.4
Dialysis (%) 2% 5% 0.1
Average HCV-RNA at HBsAb test check 3246751 3586957 0.4
Genotype 1a
Genotype 1b
Total genotype 1 (%) 80% 79% 0.9
Genoype 3 10% 10%
Other genotypes 10% 10%
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The demographics of the non-responder group were similar to the responders, with males comprising 65% and 69% of the cohort, respectively (p = 0.4). The median age at the time of the HBsAb test check was 57 years in non-responders, compared to 54 years in responders (IQR: 49–63 and 47–59, for non-responders and responders, respectively).

Clinical variables

When comparing the clinical variables among the responders and the non-responders groups, we found no differences between the rates of alcohol use (79% vs 75%), IVDU (74% vs 72%) or Diabetes Mellitus (26% vs 22%), respectively. The proportion of smokers in both groups was similar (87% and 86% respectively). Only 5% of non-responders had end-stage renal disease and were on dialysis, compared to 2% of the responders (p = 0.1).

Notably, when assessing for the presence of cirrhosis, the rate of non-responders with cirrhosis was higher than that in the responders group (29% vs 24%), but the difference was not statistically significant (p = 0.4), (Table 2). However, on multivariate analyses that included age, gender, cirrhosis, alcohol abuse, and DM, only patients with liver cirrhosis were less likely to be reactive (OR 0.66, CI 0.44–0.94). When evaluating all cirrhotic patients who received 3 or more doses in the study, we found a response rate of 77% (n = 32/139).

A total of 393 HCV patients did not have cirrhosis in our cohort. Of those, 81% (n = 317) were responders (following any number of vaccine doses), whereas 19% (n = 76) were not (following at least 3 vaccine doses). When non-cirrhotics who received at least 3 vaccine doses in both arms were assessed, the response rate was found to be 51% (79/155).

Virus-related variables

The average HCV viral load at the time of HBsAb test check among both of the groups was similar (3.5 x106 vs 3.2x106 IU/mL, for responders and non-responders respectively, p = 0.4). Both of the subgroups also had similar proportions of different HCV genotypes (Table 2).

Notably, all the above clinical and virus-related variables were also compared between responders who received 3 or more vaccine doses and non-responders, with the comparison yielding similar results (Table 3).

Table 3. Demographics, clinical and virus related-variables for responders who received ≥3 doses vs non-responders.

Variable Responders (N = 107) Non-responders (N = 107) P value
Average age at the time of HBsAb test 51 51 1
Males (%) 67% 65% 0.7
Females (%) 33% 35% 0.7
Average number of doses in those with ≧3 doses 3.5 3.6 0.5
Presence of cirrhosis (%) 26% 29% 0.6
Alcohol use (%) 94% 75% <0.0001
Tobacco use (%) 91% 86% 0.3
IV drug use (%) 72% 72% 0.6
Diabetes (%) 26% 22% 0.5
Dialysis (%) 2% 5% 0.3
Average HCV-RNA at HBsAb check 3220061 3586957 0.5
Genotype 1a
Genotype 1b
Total genotype 1 82 79 0.26
Genotype 3 13 11
Other genotypes 5 10
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Multivariate analysis

On multivariate analysis that included age, gender, cirrhosis, alcohol abuse, and DM, patients with liver cirrhosis were less likely to be re-active (OR 0.6, CI 0.44–0.94). Gender, age, alcohol abuse and DM did not show a significant correlation with HBV vaccine reactivity. These variables have been suggested before to correlate with incomplete response to vaccination [21,22].

Discussion

To our knowledge, this is one of the largest studies to date to assess HBV vaccine responsiveness in HCV infected individuals, including both clinical and virus-related variables, and various numbers of vaccine doses. We found a significantly lower response to HBV vaccination in HCV-infected individuals, with an overall response rate of 79%, and a response rate of 50% when the cohort of those who received 3 or more vaccine doses was assessed. This is significantly lower when compared to the response rate of 90% to 98% reported in the general population [11,23]. Previous reports have found similar results, with sub-optimal to significantly lower response rates in HCV patients [24,25]. A rapid decline in the proportion of patients with protective levels of HBsAb over 1 year has also been reported in this population [26].

Interestingly, a number of studies found the HBV vaccine response rate to be similar regardless of HCV status [27,28]. In a recent meta-analysis of 11 studies involving 704 HCV patients, Liu et al concluded that chronic HCV infection can decrease the immune response to a standard schedule of hepatitis B vaccination [29]. Our study validates this finding with the advantage of having a similar number of patients within one study.

The cause for the lower vaccine response in HCV-infected individuals appears to be multifactorial. HCV patients who are HBV vaccine non-responders were found to have up-regulation of PD-1, a negative immune-modulator that correlates with diminished T cell activation in response to both general and virus-specific stimulation [15]. In a murine model, HCV core has been found to strongly inhibit the cytotoxic immune response [30,31]. Lastly, impairment in the humoral arm of the immune system has been noted in HCV patients and implicated in their poor HBV vaccine response [16]. Interestingly, some reports also suggest that HBV vaccine non-responders exhibit poor responses to tetanus toxoid and Candida as well [32].

Overall, the presence of these immune changes in HCV-infected individuals likely plays a large role in vaccine hypo-responsiveness, and the extent to which immune response is decreased could be a factor related to the discrepancy between some of the studies. Genetic variation could be also related to the discrepancy in vaccine response, with genetically determined low-responsiveness to HBsAg being reported in carriers of different HLA types such as B8, B44, DR3, DR7, and DQ2, however, this is less likely to be a major contributor to the response discrepancy among HCV patients [33,34].

In the vaccine non-responder population of our study there was a trend of no-response associated to the presence of cirrhosis, which was not significant on univariate analysis, but became evident on multivariate analysis. It has been reported that cirrhosis is associated with immune dysfunction involving both B-memory-cells, and T-cells, including helper T-cells, both of which are essential in mounting immunity to HBV vaccine [16,35]. Indeed, cirrhosis has been implicated in poor responsiveness to HBV vaccine in previous reports [36,37]. Similar to other studies, we found that clinical variables including age, gender, alcohol use, IV drug use and smoking were not significantly different between responders and non-responders [17,23]. DM was found to be a poor predictor for HBV vaccine response in patients with Chronic kidney disease (CKD) and was associated with lower response to both HAV and HBV vaccines when co-present with both HCV and fibrosis [38,39]. However, our study found similar rates of Diabetes Mellitus (DM) among responders and non-responders.

Virus-related variables such as HCV genotype and viral load have been shown to play a role in HBV vaccine responsiveness. One study showed a worse response to HBV vaccine in patients who had been infected by genotype-1 of HCV as compared to those infected by genotype 2 or 3, and some manuscripts have reported an inverse correlation between increased HCV viral load and response to HBV vaccine [18,22]. However, our study found no negative impact by HCV viral load or genotype on response to vaccination. This finding may be further suggested by reports noting no increase in HBV vaccine sero-conversion following HCV treatment with achievement of sustained virological response [26].

There is no current consensus regarding a differential HBV vaccination regimen in HCV patients. In our study, some vaccine non-responders seemed to develop sero-conversion with additional dose administration. Other vaccination regimens have been previously suggested in the literature. Monkari et al. achieved a higher response rate with double dose HBV vaccine in HCV patients compared to the standard dose, with 100% of their patients responding to 40 μgs administered at the standard 0, 1, 6 month regimen [40]. In the current age of DAAs however, re-vaccination for HBV post DAA treatment seems a sensible option to achieve maximum success.

There are several limitations to our study. Firstly, it is retrospective in nature. We did not characterize cirrhotic patients by MELD or Child-Pugh scores or diabetics by glycemic control, limiting our assessment of the impact in advanced stages of these disorders. Additionally, while a large proportion of our patients received over 3 vaccines to achieve sero-conversion, we did not assess the timeline during which extra doses were administered, and subsequently any possible variation in response rates based on early vs late administration.

In conclusion, we found that HCV infection significantly impairs HBV vaccine response, with liver cirrhosis being the main risk factor for vaccine hypo-responsiveness. We found no significant association of other clinical variables or virus-related variables with vaccine response rates. Prospective studies with HBV vaccination following DAA treatment are warranted.

Supporting information

S1 File

(XLSX)

Click here for additional data file. (61.5KB, xlsx)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

JD is supported by the Robert Wood Johnson Foundation, Harold Amos Medical Faculty Development Program(AMFDP) and National Institute of Health (NIH) -NCI R21 CA215883-01A1. URLs: Robert Wood Johnson Foundation: www.rwjf.org Harold Amos Medical Faculty Development Program: www.amfdp.org/ National institute of health: www.nih.gov The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Jason Blackard

9 Apr 2020

PONE-D-20-06696

Response to hepatitis B virus vaccination in individuals with chronic hepatitis C virus infection

PLOS ONE

Dear Dr. Ashhab,

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Additional Editor Comments (if provided):

This is a retrospective analysis of HBV vaccine efficacy in HCV co-infected individuals in the US.

A relatively conservative estimate of HBV vaccine protectiveness was used.  What if that cutoff were increased to 100?

Were the sociodemographic or clinical characteristics of those that were checked for anti-HBs different from those that were not checked? (regardless of the findings)

What was the HIV status of the individuals in the responder versus non-responder groups?

How were alcohol and intravenous drug use defined?  Self-report?  Ever / never?

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: No

Reviewer #2: No

**********

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: No

**********

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Reviewer #1: Yes

Reviewer #2: No

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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: This manuscript describes the results of a retrospective evaluation of HB vaccine response in a large group of HCV infected patients in Hennepin County, MN. The authors conclude that HCV infection impairs HBV vaccine response rates.

Specific Comments

1. Page 5: The sentence regarding vaccination with Euvax B is very confusing. This vaccine is not licensed in the U.S. and the next sentence describes other vaccine products used.

2. I disagree with defining vaccine non-responders as those that received at least 1 dose. While this may be the definition in an intent to treat trial, we know from prospective registration trials that the rate of response from those who received only 1 dose is quite low and 2 doses is suboptimal. In the original registration trials from Szumness, the post-vaccine response (defined as >10 mIU/ml) was around 30% and this went up to around 70% after the second dose. In prospective trials the testing was done at short time intervals while your testing was randomly done, also decreasing apparent outcomes. At the minimum your study should focus on those who completed a full series.

3. Page 6: Your definitions of cirrhosis are very non-specific. Why not apply FIB-4 which is imperfect but is at least standardized in HCV positive patients and call cirrhosis those with FIB-4 >3.25? Use of mildly increased bilirubin in the definition allows inclusion of those with Gilbert's gene polymorphisms.

4. Page 6: Only a subset of patients had testing for surface antibody after vaccination. How did these differ from the non-tested population?

5. Page 8: Comments about genetic variation not germain to your results. Indeed, with general population rates of response of >95% to 3 doses of standard vaccine, it is hard to argue that there is much effect at all due to genetic variation.

6. Would like to sub-analysis of biopsy proven cirrhotics only before concluding that cirrhosis does not make a difference. The overall data is suspect because carefully done prospective registration trials clearly show factors like DM and dialysis clearly impact seroprotection rates. The recent report by Jackson et al (Vaccine, 2019) shows that only 65.1% of subjects receiving Engerix-B developed a response after a full series.

7. Table 1: Confusing to read. I had to calculate the number of patients in each group. Would put N for each group

8. Disagree with comparisons using 2 dose recipients. Would focus on 3 dose only for reasons described above.

9. Data in Table 3 shows 3 doses matter. Would focus on that finding though it is not a surprise.

10. Would use multivariable statistical methods to assess impact of individual factors, not just univariate methods.

Reviewer #2: This manuscript summarizes a study of HBC vaccination among patients diagnosed with HCV. This is an important issue because treatment for HCV can reactivate HBV. The study evaluated patients of a Midwest health care system who were infected with HCV and vaccinated against HBV.

Major issue:

Throughout the paper it is unclear if HBV vaccination was initiated after HCV infection occurred.

Clarity on this point is needed throughout the paper.

Some results are not consistent internally.

Specific comments:

Note: because there are no line numbers I placed quotes to find section discussed.

In general, check grammar. In particular, check capitalized words.

Introduction

Estimates of HBV and HCV prevalence utilize old estimates. The improved estimates can be found at the WHO website:

https://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/

Methods

“study, conducted” the comma should be removed

It would read better if the single sentence first paragraph was edited into two or more sentences.

Records that were available were reviewed. Whose records are not electronically available? About how many? Note: this information doesn’t need to be added to the manuscript if the number is extremely small (e.g., <5).

“Clinical information of the patients were collected” – consider beginning a new paragraph.

“Hepatitis B Surface Antibody (HBsAb)” add the word “test” or when blood drawn.

“Differences between continuous variables were evaluated at mean, using a T-Test for statistical significance.” The t-test compares the means. This sentence needs to be rewritten. Also, if you are using a t-test then it would help to provide the means and stardard deviations.

Results

“(n=1276) received various doses of HBV vaccination, 50% (n=646) of which were checked for HBsAb afterwards. Among those, 65% (n=418) developed HBsAb after vaccination (vaccine responders) and 35% (n=228) did not (vaccine non-responders).” This information is inconsistent with what is in Table 1. Specifically, in Table 1 there has 185 vaccine responders.

The other confusing issue is when patients received HBV vaccine. The methods suggests that only those who were vaccinated after HCV diagnosis are studied, however this isn’t clear throughout the paper.

A flow chart would greatly enhance the reader’s understanding of the data. The flow chart could also clarify if the HBV vaccines started before or after HCV diagnosis. Did you exclude patients who were vaccinated for HBV before HCV diagnosis? If so, providing this information on a flow chart would help the readers.

Tables in general would benefit from changing the percentages direction. The question of interest is: What proportion of those with risk factor X are responders?

Table 1 would also benefit from the addition of a line for patients who have been vaccinated according to the guidelines.

“median age at the time of the HBsAb check was 54 years in both of the groups (IQR: 47-59 and 46-50 for responders and non-responders respectively).” This can’t be correct for non-responders.

Comparison of patients who had two HBV vaccines lacks logic. The comparisons should be for patients who were vaccinated according to guidelines. You noted a difference between 2 and 3+ doses.

It would help if the analyses of risk factors are adjusted by age and sex. If age is put into categories then this adjusted analyses could still be conducted with PROC FREQ. This is particularly important for analysis of cirrhosis given that these individuals will be skewed toward older ages.

Table 3 is not helpful. It brings into the analysis patients who obtained one HBV vaccine.

Discussion

Recommend removing “largest” study given that there are likely larger studies.

The word “decreased” suggests a change. It may be more accurate to say “lower”.

In the discussion there is suggestion that patients were HBV vaccinated after HCV diagnosis. When vaccines occurred needs to be clear throughout the paper.

Overall, the discussion is well written. It is difficult to fully review the discussion section until the results are clarified.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 26;15(8):e0237398. doi: 10.1371/journal.pone.0237398.r002

Author response to Decision Letter 0

28 May 2020

The authors appreciate the reviewers’ thoughtful and insightful comments, as well as the time and effort spent. We have changed the manuscript accordingly, and revised several of the analyses per the reviewers’ requests. Below, we provide responses to each comment (please note that page and line refer to the marked-up version).

Reviewer #1:

1. Comment: “Page 5: The sentence regarding vaccination with Euvax B is very confusing. This vaccine is not licensed in the U.S. and the next sentence describes other vaccine products used.”

Response: The comment about Euvax has been removed, page 6, line 127.

2. Comment: “I disagree with defining vaccine non-responders as those that received at least 1 dose. While this may be the definition in an intent to treat trial, we know from prospective registration trials that the rate of response from those who received only 1 dose is quite low and 2 doses is suboptimal. In the original registration trials from Szumness, the post-vaccine response (defined as >10 mIU/ml) was around 30% and this went up to around 70% after the second dose. In prospective trials the testing was done at short time intervals while your testing was randomly done, also decreasing apparent outcomes. At the minimum your study should focus on those who completed a full series.”

Response: We have updated our definition of non-responders to that of those receiving 3 or more doses of the vaccine. We have updated all the comparisons and tables accordingly. That has led us to remove some of the data (such as average number of vaccine doses in non-responders) and to re-calculate some analyses, however, for the most part, the results of the study remained the same. Table 2 now contains comparisons between all responders to non-responders (page 19), and table 3 (page 20) now contains comparisons between responders with 3 or more doses, to non-responders. Any previous data related to comparisons involving non-responders with less than 3 vaccine doses were removed from the manuscript. We also added results from Multivariate analysis, again, with non-responders being modified to only those who received 3 or more doses (page 9, lines 204-207).

3. Comment “Page 6: Your definitions of cirrhosis are very non-specific. Why not apply FIB-4 which is imperfect but is at least standardized in HCV positive patients and call cirrhosis those with FIB-4 >3.25? Use of mildly increased bilirubin in the definition allows inclusion of those with Gilbert's gene polymorphisms.”

Response: Our definition of cirrhosis included increased INR or increased bilirubin plus low platelet count AND cirrhotic looking liver (hence a combination of imaging and laboratory variables, which are recommended by the European Association for the Study of the Liver as referenced) or a combination of 3 laboratory parameters (two of coagulopathy and one of internal liver physiology). We believe there has been a confusion, and we have changed the wording for better clarification (page 6, lines 135-140). FIB4 and APRI scores have been reported to have a potential use for fibrosis assessment, but no international hepatology guideline supports them for cirrhosis diagnosis.

4. Comment “Page 6: Only a subset of patients had testing for surface antibody after vaccination. How did these differ from the non-tested population?”

Response: Demographics showed that those not tested for HBsAb were 63% male compared to 70% male in those tested, 14% of those not tested had cirrhosis versus 25% of those tested (this yielded a significant difference, p=0.01). Mean ages were similar between both groups: 48 y/o (CI 47-50) vs tested 54 y/o (CI 53-54), p=0.001). We have added this information to the manuscript (Page 7, line 159-163).

5. Comment “Page 8: Comments about genetic variation not germain to your results. Indeed, with general population rates of response of >95% to 3 doses of standard vaccine, it is hard to argue that there is much effect at all due to genetic variation.”

Response: We have placed references that studied genetic variations (references 32-33). However, we understand the comment as a practical point. We have added a comment indicating the low likelihood of this playing a role (we believe it is worth mentioning at least). Page 10, lines 237-238.

6. Comment “Would like to sub-analysis of biopsy proven cirrhotics only before concluding that cirrhosis does not make a difference. The overall data is suspect because carefully done prospective registration trials clearly show factors like DM and dialysis clearly impact seroprotection rates. The recent report by Jackson et al (Vaccine, 2019) shows that only 65.1% of subjects receiving Engerix-B developed a response after a full series.”

Response: Our analysis is different than that quoted by the reviewer. The reviewer quotes a study in which 65% of cirrhotics respond to HBV vaccine. We mention that among our responders there were 24% cirrhotics, these are different ways of looking at data. We have looked at the response rate among cirrhotics that got 3 doses of the vaccine, and found a response rate of 77%. This was added to the body of the study (Page 8, lines 188-193).

7. Table 1: Confusing to read. I had to calculate the number of patients in each group. Would put N for each group

Response: Table 1 was modified as requested. Numbers of patients that received any number of doses was added (Table 1, Page 18).

8. Disagree with comparisons using 2 dose recipients. Would focus on 3 dose only for reasons described above.

Response: We re-performed the analysis, assessing all responders vs non-responders with 3 or more doses (Table 2, Page 19) and Responders with 3 or more doses compared to non-responders with 3 or more doses (table 3, page 20). The “results” section, page 7, was re-written to indicate those changes and the numbers were updated.

9. Data in Table 3 shows 3 doses matter. Would focus on that finding though it is not a surprise.

Response: Similarly, as above, we have re-done the analysis with 3 doses and modified the table accordingly, as above.

10. Would use multivariable statistical methods to assess impact of individual factors, not just univariate methods.

Response: We have now included multivariate analysis to our study. On multivariate analysis we found that cirrhosis correlated with less reactivity compared to those without cirrhosis (OR 0.66, CI 0.44-0.94). In the multivariate analyses, alcohol abuse and diabetes did not show a significant correlation between HBsAb reactivity. This was added to the body of the text (Pages 9, lines 204-207).

Reviewer #2:

-Comment 1: “Throughout the paper it is unclear if HBV vaccination was initiated after HCV infection occurred. Clarity on this point is needed throughout the paper”.

Response: As the reviewer can imagine it is certainly not possible to know when patients got infected with HCV, as the great majority of infections are subclinical, we can at best estimate based on risk factor timing. To provide an estimation for the reviewer, we did assessed the percentage of first HBV vaccinations in relation to the first positive test for HCV. From a small subset of individuals (255) in which we assessed this information, we found that 177 had the first vaccination before being tested for hepatitis C. It should be understood however that the great majority of individuals expressed a remoted risk factor for HCV infection, with likely linking of risk-factor to infection at least 1-2 decades before laboratory check. This was added to the methods section, Page 6, lines 129-132.

-Comment 2:

“Estimates of HBV and HCV prevalence utilize old estimates. The improved estimates can be found at the WHO website:

https://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/”

Response: We have added new estimates per the reviewer’s request (Page 4, lines 76-77).

-Comment 3: “study, conducted” the comma should be removed

Response: comma removed (Page 5, line 104).

-Comment 4: “It would read better if the single sentence first paragraph was edited into two or more sentences.”

Response: The paragraph was edited into 2 sentences (Page 5, lines 104-107)

-Comment 5:” Records that were available were reviewed. Whose records are not electronically available? About how many? Note: this information doesn’t need to be added to the manuscript if the number is extremely small (e.g., <5)”.

Response: Quote “all available” just means that all the records that we had were reviewed. We changed the paragraph per reviewers request (Page 5, line 108).

-Comment 6: “Clinical information of the patients were collected” – consider beginning a new paragraph.

Response: The sentence was changed to a new paragraph (Page 5, line 113).

-Comment 7: “Hepatitis B Surface Antibody (HBsAb)” add the word “test” or when blood drawn.

Response: We added “test” following HBsAb throughout the study.

-Comment 8: “Differences between continuous variables were evaluated at mean, using a T-Test for statistical significance.” The t-test compares the means. This sentence needs to be rewritten. Also, if you are using a t-test then it would help to provide the means and standard deviations.

Response: We have changed the wording of the phrase after talking to our Statistitian to: “Statistical analyses were performed using SAS version 9.4. Differences between continuous variables were evaluated at the means using a T-Test. Categorical variables were evaluated using a Chi-Square test. The multivariate regression was modeled as a logistic regression.” This is reflected on Page 6, lines 147-149.

-Comment 9: “(n=1276) received various doses of HBV vaccination, 50% (n=646) of which were checked for HBsAb afterwards. Among those, 65% (n=418) developed HBsAb after vaccination (vaccine responders) and 35% (n=228) did not (vaccine non-responders).” This information is inconsistent with what is in Table 1. Specifically, in Table 1 there has 185 vaccine responders.

Response: The 185 patients noted as responders in table 1 are the responders with known number and timing of doses. The 418 responders noted in the text are all patients with documented vaccination in the EMR, but not all had available timing and number of doses.

Only the 185 patients were included in analyses pertaining to number of doses, however the remainder of patients were included in analyses regarding clinical and virus related variables, as these were available. Notably, we also re-did all the analyses for those with known 3 or more doses in the responder arm, compared to non-responders (table 3, page 20) and that yielded similar results when compared to the analyses in table 2, page 19. This was also clarified in the results section of the study, page 8, lines 200-202.

-Comment 10: ‘The other confusing issue is when patients received HBV vaccine. The methods suggests: that only those who were vaccinated after HCV diagnosis are studied, however this isn’t clear throughout the paper.”

Response: This point was address above. We have indicated the number of individuals that received vaccination before and after testing positive to clarify this point, page 6, lines 129-132.

-Comment 11: “-A flow chart would greatly enhance the reader’s understanding of the data. The flow chart could also clarify if the HBV vaccines started before or after HCV diagnosis. Did you exclude patients who were vaccinated for HBV before HCV diagnosis? If so, providing this information on a flow chart would help the readers.”

Response: A flow chart was added (Page 17, figure 1).

-Comment 12: “Tables in general would benefit from changing the percentages direction. The question of interest is: What proportion of those with risk factor X are responders?”

Response: We appreciate the reviewers comment. However, our study is looking at which factors are associated with no response, which we believe is reflected in our tables. Changing the positioning of the tables would require changing also the analyses and reflective p values and we believe that would be a total different manuscript than the one we are submitting. We have however, considerably modified the tables based on the re-analysis.

-Comment 13: “Table 1 would also benefit from the addition of a line for patients who have been vaccinated according to the guidelines.”

Response: All patients with 3 doses were vaccinated per the guidelines. Those in the study who had interrupted timings restarted the 3 dose sequence and ended up receiving 4 or more doses.

-Comment 14 “median age at the time of the HBsAb check was 54 years in both of the groups (IQR: 47-59 and 46-50 for responders and non-responders respectively).” This can’t be correct for non-responders.

Response: The age analysis was redone to identify non-responders as those who did not respond after getting at least 3 doses. Means have now changed for non-responders (Page 8, lines 177-179).

-Comment 15: “Comparison of patients who had two HBV vaccines lacks logic. The comparisons should be for patients who were vaccinated according to guidelines. You noted a difference between 2 and 3+ doses.”

Response: Comparisons between patients with 2 doses were removed. Now the comparison is between patients who received 3 or more doses (Page 20, table 3).

-Comment 16: “It would help if the analyses of risk factors are adjusted by age and sex. If age is put into categories then this adjusted analyses could still be conducted with PROC FREQ. This is particularly important for analysis of cirrhosis given that these individuals will be skewed toward older ages.”

Response: We have performed and added a multivariate analysis and included age per reviewer’s request (Pages 9, lines 204-207).

-Comment 17: “Table 3 is not helpful. It brings into the analysis patients who obtained one HBV vaccine.”

Response: Table 3 now modified and analysis was re-done to include patients who received 3 or more vaccines only (Page 20, table 3).

-Comment 18: “Recommend removing “largest” study given that there are likely larger studies.”

Response: Through our literature review, we have not encountered a study of a similar size, aside from the noted meta-analysis. However, the phrase was substituted with “one of the largest studies”, per the reviewer’s request (Page 9, line 210).

-Comment 19: “The word “decreased” suggests a change. It may be more accurate to say “lower”.

Response: The word “Decreased” was switched to lower, when pertaining to the response to the vaccine, throughout the study.

-Comment 20 “In the discussion there is suggestion that patients were HBV vaccinated after HCV diagnosis. When vaccines occurred needs to be clear throughout the paper”

Response: We have added a clarification on the percentage of patients that received vaccination before or after diagnosis. We would like to emphasize that this really does not correlate with “hepatitis C acquisition” as that is not possible to define, since most infections are subclinical. Clarification was added to the “methods”, page 6, lines 129-132.

Additional comments:

-A relatively conservative estimate of HBV vaccine protectiveness was used. What if that cutoff were increased to 100?

Response: Our cut off (a titer of 12 mIU/mL) is above the recommended cut off of 10 mIU/mL, deemed to be protective against hepatitis B (West DJ, Calandra GB. Vaccine induced immunologic memory for hepatitis B surface antigen: implications for policy on booster vaccination. Vaccine. 1996;14(11):1019‐1027.), which is also the cut off recommended by the CDC.

-Were the sociodemographic or clinical characteristics of those that were checked for anti-HBs different from those that were not checked? (Regardless of the findings)

Response: We assessed the characteristics of those who were tested to document immunity and those who were not, patients who were not tested were younger, both groups were predominantly male, this data was added (Page 7, Lines 159-163).

-What was the HIV status of the individuals in the responder versus non-responder groups?

Response: Because of the low number of individuals with HIV (<2%) in the population, these individuals were removed from the study.

- How were alcohol and intravenous drug use defined? Self-report? Ever / never?

Response: History of alcohol abuse, was obtained from the chart, by assessing for documentation of use compatible with abuse or for a diagnosis of alcohol abuse, related to recurrent alcohol related presentations and positive toxicology in those who did not self-report heavy use. To ensure the patients had active alcohol abuse at the time of vaccination, only patients who were noted to have ongoing abuse in the hepatology clinic were defined as abusers for the purpose of the study.

IV drug use was obtained from EMR charts and problem lists that could have been a consequence of previous positive toxicology or relied on self-report. Patients were considered to have IV drug abuse if the evaluating provider noted a history of active or recent drug use on presentation to the hepatology clinic.

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Decision Letter 1

Jason Blackard

23 Jun 2020

PONE-D-20-06696R1

Response to hepatitis B virus vaccination in individuals with chronic hepatitis C virus infection

PLOS ONE

Dear Dr. Ashhab,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

Reviewer 1 remains unconvinced of the methods and findings.  Please address the comments from reviewer 1 thoroughly in a revised manuscript.

Please submit your revised manuscript by Aug 07 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Jason Blackard, PhD

Academic Editor

PLOS ONE

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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: (No Response)

Reviewer #2: All comments have been addressed

**********

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: No

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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: No

Reviewer #2: No

**********

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

**********

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: This revised manuscript provides a retrospective analysis of the HBV vaccine response rates to a large number of HCV infected persons. The authors primary conclusion is that that cirrhosis is a key factor in failure to respond.

Specific Comments

1. Abstract: The authors conclude that HCV infection impairs HBV vaccine response with cirrhosis being the only identifiable risk factor. This conclusion is not supported by the data presented. There is no evidence that HCV specifically impairs HBV vaccine response. Indeed, the statement actually says that cirrhosis is the factor that impairs response (among those with HCV). This distinction is important.

2. Page 7: I remain confused about the numbers and calculations. The rate of response in those that received 3 doses was 42%. Then the authors note that 77% of those that received 3 or more doses had a response but in parentheses it says (32/139). This is a 23% response rate.

3. The overall response rate in non-cirrhotic patients who completed 3 or more doses is not explicitly stated. However, the overall rate of response among those who received 3 or more doses was 79.5%.

4. In univariate analysis there was no difference in response related to cirrhosis with the proportions of cirrhotics among responders and non-responders essentially equal (p= 0.6). To then select a subset of characteristics and find significance for cirrhosis is a bit of statistical gamesmanship.

5. Page 7: After stating that 418/525 (79.6%) have a vaccine response, the next sentence says "Out of all who receive 3 or more doses only 50% develop HBsAb.

Reviewer #2: All comments were sufficiently addressed.

The data is not currently available. However, the authors state they will make the data available upon publication. This meets the goal of the requirement.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 26;15(8):e0237398. doi: 10.1371/journal.pone.0237398.r004

Author response to Decision Letter 1

7 Jul 2020

The authors appreciate the reviewer’s thoughtful and insightful comments, as well as the time and effort spent.

1. Abstract: The authors conclude that HCV infection impairs HBV vaccine response with cirrhosis being the only identifiable risk factor. This conclusion is not supported by the data presented. There is no evidence that HCV specifically impairs HBV vaccine response. Indeed, the statement actually says that cirrhosis is the factor that impairs response (among those with HCV). This distinction is important.

Response: We appreciate the reviewer’s comment. Our data shows a 79% HBV vaccine response, that is much lower than the >90% reported in the general population in multiple studies, and a 50% response rate in the population who received 3 or more doses. We have changed the comment in the abstract to clarify (page 3, line 66). To further clarify this to the readers, we have added the response rates in non-cirrhotic HCV patients in our cohort, which is also well below the rate reported in the literature, to the results section (page 8, lines 188-191).

2. Page 7: I remain confused about the numbers and calculations. The rate of response in those that received 3 doses was 42%. Then the authors note that 77% of those that received 3 or more doses had a response but in parentheses it says (32/139). This is a 23% response rate.

Response: We apologize for the confusion regarding the second portion of the comment, we made a typo (32 were non responders, but 107 were, accounting for a response rate of 77% as indicated). We have fixed this issue in the manuscript (page 8, line 187). We have to emphasize however, that as indicated in that line, this is the response rate in cirrhotics who received 3 or more doses. When assessing the entire population who received 3 or more doses, is detailed in table 3, page 20, response rate is 50% (equal number of patients in each arm).

* Clarification: The “42%” noted in table 1, is not the response rate, but rather, the proportion of responders, who sero-converted following 3 doses of the vaccine, as is indicated in the column title.

3. The overall response rate in non-cirrhotic patients who completed 3 or more doses is not explicitly stated. However, the overall rate of response among those who received 3 or more doses was 79.5%.

Response: Thank you for the comment. As we indicated above, we have added the lines 188-191, in page 8, to clarify the response rate in non-cirrhotics only, which as indicated above, is significantly lower than the response rate in the general population.

4. In univariate analysis there was no difference in response related to cirrhosis with the proportions of cirrhotics among responders and non-responders essentially equal (p= 0.6). To then select a subset of characteristics and find significance for cirrhosis is a bit of statistical gamesmanship.

Response: We appreciate the reviewer’s comment. However, we carefully chose the characteristics (Age, gender, alcohol abuse, and DM) as each of these has been reported to be associated with decreased HBV vaccine response in the literature. We have added a comment to clarify this (Page 9, line 203). Seeing as the proportion of Cirrhosis was higher in the non-responders’ arm, but no statistically, a concern was that those characteristics may have been confounders that limited the univariate analysis. A multivariate analysis accounting for possible confounders was also suggested by reviewer 2 at the time of the initial submission.

5. Page 7: After stating that 418/525 (79.6%) have a vaccine response, the next sentence says "Out of all who receive 3 or more doses only 50% develop HBsAb.

Response: Our initial statement says “various doses” indicating that we are including patients that responded even with 2 doses of the vaccine. The statement includes those that received all 3 doses. We have changed “various” to “any number” of HBV vaccine doses to make that more clear (page 7, line 161-162).

Attachment

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Decision Letter 2

Jason Blackard

27 Jul 2020

Response to hepatitis B virus vaccination in individuals with chronic hepatitis C virus infection

PONE-D-20-06696R2

Dear Dr. Ashhab,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jason Blackard, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

None

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: Yes

Reviewer #2: (No Response)

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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: No

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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: Revised manuscript addresses key issues.

Might want to include 95% C.I. of overall response rate among HCV positive (78-84%) which does indeed support rate being lower than non-HCV healthy population.

Reviewer #2: The authors will make data available upon publication. This is acceptable.

Only style changes I recommended previously were not made. This is ok.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Acceptance letter

Jason Blackard

13 Aug 2020

PONE-D-20-06696R2

Response to hepatitis B virus vaccination in individuals with chronic hepatitis C virus infection

Dear Dr. Ashhab:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jason Blackard

Academic Editor

PLOS ONE

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