Background: There is currently no virological cure for chronic HBV infection. In Hepatitis B e antigen negative hepatitis, the goal of therapy is hepatitis B surface antigen loss, which is rarely achieved. Stopping treatment with nucleos(t)ide analogues (NAs) in e negative hepatitis often leads to viral relapse. So, most patients need lifelong therapy. Lifelong therapy has many limitations including cost of therapy compliance, and adverse event like renal and bone toxicity, viral mutant and drug resistance. A few recent studies have shown that finite treatment is an option in a subset of e negative chronic hepatitis. However clinical experience is still restricted in a few parts of the world and this renders the decisions quite challenging for the physicians. Regarding possible predictors of post-NA outcomes, different studies have evaluated various factors, which can be categorized as markers of hepatitis B virus activity, markers of host immune response, and markers of other patient characteristics. In clinical practice, the most important question for patients who discontinue NAs is to differentiate those who will benefit by achieving HBs Ag loss or at least by remaining in remission and those who will relapse requiring retreatment. Most of the therapy discontinuation studies so far came from East Asian and only a few from European population and examined the rates and predictors of post-NA virological and/or combined relapses or HBs Ag loss. To date, there is still controversy about predictors of post-NA relapses, while only serum HBs Ag level at NA discontinuation appears to be a robust predictive marker of the probability of subsequent off treatment HBs Ag seroclearance. So there is an unmet need to identify off therapy predictor of response.

Hypothesis: Long term suppression of the viremia may revert T cell exhaustion and restore immune control. Stopping NA treatment and the subsequent sudden increase in HBV DNA and HBV proteins may also be an important trigger to re-induce T-cell responses against HBV-infected hepatocytes. This may explain the loss of HBsAg in a subset of e negative chronic hepatitis patients and may lead to a functional cure.

Aims:

a) Off therapy virological and clinical response at 6,12,18,24 months following cessation of oral antiviral in e negative non-cirrhotic chronic viral hepatitis patients. 

b) Evaluation of HBs Ag loss and change in HBs Ag titer in patients who stop antiviral compared to patients who continue antiviral therapy at 24 months

c) Off therapy Immunological changes with effects on CD8+ T cells, CD4+ T cells and different serum cytokine level.

Method: Non cirrhotic e negative chronic hepatitis B patients who received NA for more than 2 years and achieved virological and biochemical response will be recruited and randomized into either therapy stop group or continuing therapy group. The study will recruit 40 non-cirrhotic e negative hepatitis patients, 20 patients in arm A, who will stop oral anti-viral (TDF/ETV), and 20 patients in arm B who will continue antiviral monotherapy. All patients will be followed up for 24 months at regular intervals of 3 months for ALT and for DNA every 6 monthly.

Measurement of following virological and immunological parameters will be carried out - serum HBs Ag, serum HBV DNA, ALT, anti-HBs, T cell, and levels of different serum cytokines. The endpoint of the study will be a virological relapse or clinical relapse or end of the study duration.

Virological relapse will be defined if HBV DNA increased to10,000 copies/ml.

Clinical relapse will be defined if ALT increased to 2 x UNL along with HBV DNA more than 10,000 copies/ml.

Statistics:

Continuous data were indicated with mean ± SD while categorical data were reported as number and percentage (%).

Spearman correlation coefficient was used to investigate the correlation among HBs Ag, HBV DNA, and T lymphocytes.

Nonparametric tests including Mann–Whitney test and Wilcoxon signed-rank test were used to compare means between groups for data normality was not assumed

Wilcoxon rank-sum test will be used to compare the change in virological and biochemical and immunological parameters along with a median decline in HBs Ag from the baseline between patients who stopped oral antiviral and patients who continued on oral antiviral.

The sample size will be calculated using n Master 2.0 software.

It is assumed within the study period without therapy 25% of patients may have relapsed and with therapy, 5% of patients may relapse. With this assumption, each group will need 16 patients but we will recruit 20 patients in each group as there will be some drop out during study period.

Timeline: During the first three months, 40 e negative non-cirrhotic patients will be recruited from the Hepatology and NVHCP clinic of our Institute, IPGME&R, Kolkata, India. After randomization patients will be followed up for 2 years.

PROJECT PROPOSAL

Background: Asia-Pacific region contributes more than half of the global population. In 2015 the Asia-pacific region contributed 60% of the global liver-related death [1]. In this region, HBV contributes more than 50% cirrhosis related death and 50% HCC related death [1]. One of the most cost-effective ways to battle this deadly virus is universal vaccination. But vaccine coverage in many countries in the Asia-Pacific region is not adequate. Moreover, many patients are already infected and need treatment. WHO has set up a goal to decrease mortality and morbidity related to chronic viral hepatitis. By 2030 we need to identify and treat at least 80% of treatment eligible HBV infected patients [2].To achieve these goals, recently the government of India has launched the National Viral Hepatitis Control Program which would upscale screening and treatment of chronic viral hepatitis. This Program will bear the entire cost of treatment. Currently, there is no virological cure for Hepatitis B, but successful nucleotide analog therapy can suppress hepatitis B virus DNA replication. Stopping nucleotides analog (NA) therapy often leads to viral relapse and so lifelong therapy is usually required [3]. In HBe Ag-negative disease, the goal of therapy is hepatitis B surface antigen (HBs Ag) loss [4]. However, HBs Ag loss is a very rare event, so most of the patients need lifelong therapy which is not only expensive and also put the patients at risk of drug-related complications. In long term therapy, compliance is often poor and irregular therapy can cause drug resistance. Life-long therapy will also add a huge financial burden to the National Viral Hepatitis Control Program. Contradictory to lifelong therapy, recent studies have reported that after the long term suppression of HBV, anti-viral therapy can be discontinued and after cessation of antiviral, many patients maintain virological response [5]. Chang et al. conducted a systemic review in which twenty-two studies with a total of 1732 patients were identified and included [6]. The median duration of therapy, consolidation therapy and off-therapy follow- up ranged from 6 months to 8 years, 4 to 96 weeks, and 6 to 80 months respectively. Patients were monitored with serum ALT and HBV DNA monthly in the first 1–3 months and every 3–6 months afterward in most studies. The 1-year off-therapy ‘virological relapse’ (HBV DNA >2000 IU/mL) and ‘clinical relapse’ (HBV DNA > 2000 IU/mL + ALT elevation) occurred in 70% and 50% of the patients, respectively, and <40% of the patients received re-treatment. Off-therapy severe flares were rare and hepatic decompensation was reported in only one patient with cirrhosis. Biochemical relapse reflecting enhanced immune-mediated hepatocyte killing may lead to a higher chance for off-therapy HBs Ag seroclearance67 and be possibly desirable. But most of the data in this population are based on retrospective studies with a limited duration of NA therapy. Most of the studies are in Chinese patients (predominant HBV genotype B/C) or in European patients (dominant HBV genotype A and D) and there is great heterogeneity among the studies in terms of treatment duration, duration of virological remission before NA discontinuation, and duration of follow up after discontinuation of NA. Van Hees et al [7], had shown that Caucasian ethnicity, but not Treatment Cessation is Associated with HBs Ag loss following NA induced HBe Ag seroconversion. HönerZuSiederdissen et al [8] in their study on stopping nucleotide therapy in e negative non-cirrhotic chronic hepatitis B patients had measured immunological changes. Virological relapse was associated with induction of plasma TNF-α, IL-10, IL-12p70 and CXCL10 and subsequent decline in HBs Ag. The peak levels of HBV DNA and hepatitis B core-related antigen following cessation of therapy were positively correlated with the level of HBs Ag decline at week 48. A recent study had demonstrated that T cells of patients that subsequently achieved HBs Ag loss after NA withdrawal (3/15) were less exhausted at all-time points and displayed a higher proliferative capacity 12 weeks after treatment discontinuation [9]. Another study depicted that NK cells with less inflammatory phenotype were associated with HBs Ag seroconversion and may represent a potential marker for treatment withdrawal [10]. At present EASL and APASL guidelines suggest discontinuation of antiviral is one option in e negative non-cirrhotic chronic hepatitis provided the patient is willing for close biochemical and virological follow-up [11,12]. But none of the guidelines discuss predictors of off-therapy virological response. Analysis of the virological and immunological interplay is required to correctly elucidate the physiological mechanism of the drug withdrawal approach. So we have designed this study to comprehensively assess virological, clinical response as well as immunological changes associated with off therapy virological response so that we can predict off therapy response and compare with them with on therapy response. This study may help our National Program to adopt finite therapy in a subset of e negative hepatitis B patients and might strengthen national policy regarding the management of such patients

Hypothesis: Although the mechanism leading to immune-control after treatment withdrawal has not been fully elucidated, there is the debate whether long term suppression of the viremia may revert T cell exhaustion and restore immune control. This would mean that sudden HBV DNA increase would trigger a vigorous T cell response against HBV infected hepatocytes. This immune reconstitution scenario would be a possible explanation for the so called ‘benign’ ALT flares that precedes HBs Ag loss in these patients. Also, the clinical observation of HBs Ag clearance or virological control without hepatic flare or even persistent low HBV viremia suggests an underlying effective immune control of HBV infection with maintained suppression of HBV replication.

Aims:

The aims of the study are as follows-

• To assess off therapy virological and clinical response at 6,12,18 and 24 months following cessation of oral antiviral in Indian patients with HBe Ag negative non-cirrhotic chronic viral hepatitis.

• To evaluate HBs Ag loss and changes in HBs Ag titer in patients who stop antiviral therapy as compared to patients who continue treatment at 24 months

• To study off therapy Immunological changes with effects on CD8+T cells, CD4+T cells and different serum cytokine levels.

Approach:

Study area- Hepatology and NVHCP clinic of School of Digestive and Liver disease, IPGME& R, SSKM Hospital, Kolkata, India.

Study population-HBe Ag negative chronic hepatitis patients who had received TDF or ETV for > 2 years and achieved a biochemical and virological response.

Study period- 24 months and first 3 months will for recruitments for patients and from then we will follow for 2 years and it will have started from September 2021 to August 2023

Sample size– We plan to recruit 40 patients, 20 in arm A, who will stop oral anti-viral (TDF/ETV), and 20 in arm B who continue antiviral monotherapy.

Study design:

Prospective Randomized, single-center study. Patients will be randomly assigned 1:1 to either stop (arm A) or continue (arm B) TDF/ETV monotherapy. The random numbers will be used from Kevin Conroy: 5120 Random Numbers (<5k,2002) [Call the JavaScript pseudo-random number generator.] Website:RandomNumber.org.2004.

All patients will be followed up for 24 months. ALT will be checked every 3 months and HBs Ag titer and HBV DNA will be carried out every 6 months or when indicated.

Study Assessment-Measurement of serum HBs Ag, serum HBV DNA, ALT, anti-HBs, Fibro-scan assessment/USG Abdomen assessment/APRI/FIB4, T cells response and serum cytokines.

Serum HBs Ag quantification will be done using the Abbott Architect i1000sr platform according to the manufacturer’s instructions.

Serum HBV DNA Quantification- HBV DNA will be extracted from 200 μl serum samples using Blood Mini Kit (Qiagen Inc., CA, USA), and the quantification of HBV DNA will be performed by Real-Time PCR using SYBR Green Master Mix (Applied Bio systems) and HBV-specific primers.

Serum ALT will be measured by a modified IFCC method, kinetic technique.

Serum Anti HBs Ag will be measured by the sandwich ELISA method.

Level of different cytokines in the sera of study population will be determined by cytometric bead array (CBA), by using anti-human Th1/Th2/Th17 kit (BD Bioscience), according to the manufacturer’s protocol.

Assessment of HBV-specific T cell response- Peripheral blood mononuclear cells (PBMC) will be isolated from EDTA blood of study subjects using Histopaque density gradient centrifugation and stimulated with HBV overlapping peptides (OLP) (a set of 15-mer peptides overlapping by 10 residues spanning genotype D HBV core protein, 5μg/ml) (Mimotopes, Australia) in RPMI 1640 complete medium for 5 days at 37°C. On day 4, PBMC will be restimulated with 5μg/ml OLP overnight, in the presence of Brefeldin A (1μg/ml). To estimate the expression level of cytokines in virus-specific T cells, PBMC will be stained with anti-CD4FTIC, anti-CD8PE and then washed, fixed, and permeabilized by using BD Cytofix/Cytoperm kit and further labeled with APC-conjugated antibodies against IFN-γ, IL-2, TNF-α, and IL-10. Next the cell will be washed and analyzed by flow cytometry.

Inclusion criteria:

1.      Adult patients (age 18-65 years) with e negative chronic hepatitis B

2.      Continuous TDF or ETV monotherapy for at least 2 years

3.      HBV DNA < 250 copies/ml for at the time of enrolment

4.      Sustained viral suppression (at least 3 reports of HBV DNA at 6 months’ interval showing undetectable viral load).

Exclusion criteria:

1.      Patients with significant fibrosis or cirrhosis (fibro-scan> 8 kpa or biopsy or USG Upper Abdomen shows cirrhosis, APRI >1.5, FIB-4 >3.25)

2.      Immunocompromised

3.      HCC

4.      Pregnancy

5.      Co-infection

6.      Patients who received dual antiviral or PEG IFN

7.      Patients on lamivudine or adefovir

8.      Patient not willing to participate in the study

Study endpoint:

1. If there is a virological relapse or clinical relapse.

Virological relapse will be defined if HBV DNA increased to10,000 copies/ml.

Clinical relapse will be defined if ALT increased to 2 x UNL along with HBV DNA more than 10,000 copies/ml

 2.  If the patient reaches the end of the follow up (24 months).

Statistical analysis:

Data entered will be done in MS EXCEL Spread Sheet,

Data analysis will be carried out by SPSS version 16.0.

Continuous data were indicated with mean ± SD while categorical data were reported as number and percentage (%).

Spearman correlation coefficient was used to investigate the correlation among HBs Ag, HBV DNA, and T lymphocytes.

Nonparametric tests including Mann–Whitney test and Wilcoxon signed-rank test were used to compare means between groups for data normality was not assumed

Wilcoxon rank-sum test will be used to compare the change in virological and biochemical and immunological parameters along with a median decline in HBs Ag from the baseline between patients who stopped oral antiviral and patients who continued on oral antiviral.

All P-value <0.05 will be considered statistically significant.

Calculation of sample size: Sample size is calculated using n Master 2.0 software. It is assumed within the study period without therapy 25% of patients may have relapse and with therapy, 5% of patients may have relapse. With this assumption, each group will need 16 patients but we will recruit 20 patients in each group as there will chance of drop out during study.

                                           Flow Chart 

                                      e Antigen Negative  CHB

                                                  Randomization

                    ARM A (off therapy) 20 patients)                                                            ARM B (on therapy)  (20 patients)                                          

 Statistics