Contrasting timing of virological relapse after discontinuation of. tenofovir or entecavir in hepatitis B e antigen-negative patients.

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1 Contrasting timing of virological relapse after discontinuation of tenofovir or entecavir in hepatitis B e antigen-negative patients. Running title: Difference after stopping TDF or ETV Christoph Höner zu Siederdissen 1, Aric Josun Hui 2, Wattana Sukeepaisarnjaroen 3, Pisit Tangkijvanich 4, Wei Wen Su 5,6, Gerardo Enrique Guillén Nieto 7, Paul Gineste 8, Josianne Nitcheu 8, Sandrine Crabé 8, Sandrine Stepien 9, Michael P. Manns 1, 11, Christian Trépo 10, Heiner Wedemeyer 1, 11,12, Markus Cornberg 1, 11* 1 Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany 2 Alice Ho Miu Ling Nethersole Hospital, Hong Kong, China 3 Khon Kaen University, Khon Kaen, Thailand 4 Center of Excellence in Hepatitis and Liver Cancer, Chulalongkorn University, Bangkok, Thailand 5. Department of Gastroenterology, Changhua Christian Hospital, Changhua, Taiwan, ROC 6. School of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC 7 Centro de Ingeniería Genética y Biotecnología, Havana, Cuba 8 ABIVAX, PARIS, France 9 Georges Institute, Sydney, Australia 10 Department of Hepatology, Groupement Hospitalier Nord, Hospices Civils de Lyon, Lyon France - Lyon University, Lyon, France The Author(s) Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, journals.permissions@oup.com.

2 11 German Center for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany 12 Department of Gastroenterology and Hepatology, University Clinic Essen, Germany *Corresponding author: Markus Cornberg, Phone: Hannover Medical School, Carl-Neuberg-Str. 1, Department of Gastroenterology, Hepatology and Endocrinology, Hannover, Germany Previously presented: AASLD 2017, Wedemeyer H et al. J Hepatol 2017;66:S101. Abstract Stopping long-term nucleos(t)ide analogue therapy increases HBsAg loss rates in HBeAgnegative patients. Viral rebound may induce immune responses facilitating functional cure. We analyzed which factors are associated with timing of virological relapse in 220 Asian HBeAg-negative patients from the prospective ABX203 vaccine study. Unexpectedly, only the type of antiviral therapy was significantly associated with early virological relapse, defined as HBV DNA > 2,000 IU/ml until week 12 and occurred earlier in patients treated with tenofovir versus entecavir (median time 6 versus 24 weeks, p < ). This should be considered for future trials and monitoring of patients after treatment discontinuation. Key words: Virological relapse, nucleos(t)ide discontinuation, tenofovir, entecavir, HBeAg negative hepatitis B virus infection

3 Background: Treatment with the nucleos(t)ide analogues (NA) tenofovir or entecavir is the current mainstay of therapy in chronic hepatitis B (CHB). Whereas NA therapy can be discontinued in some HBeAg positive patients after seroconversion, current guidelines usually recommend treatment until HBsAg seroclearance in HBeAg-negative patients (1,2). The concept of earlier treatment discontinuation in HBeAg-negative patients with CHB has been implemented first in the APASL guidelines and has also been suggested in other current international guidelines (1,2). Although, one third of patients remain in virological remission, relapse is common in the first months after treatment discontinuation (3). Several studies have shown that treatment discontinuation in HBeAg-negative CHB can foster HBsAg decline and even HBsAg loss in some patients presumably by inducing immune responses (4 7). We showed that the peak of HBV DNA rebound until week 12 after stopping treatment was associated with the subsequent decline of HBsAg (5). However, optimal timing of reinitiating antiviral therapy in case of viral relapse is not well defined. Thus, early virological relapse may be of particular importance for the induction of an immune response against HBV, which could have beneficial long-term effects. Here we took advantage of the availability of data derived from the largest prospective trial that analyzed relapse after stopping NA therapies. We aimed to assess factors associated with the timing of virological relapse. Methods Data of the present study were derived from the ABX203 study. The ABX203 study is a randomized multi-center study, investigating whether ABX203 (developed as NASVAC by CIGB, Cuba) influences the risk for virological relapse in patients after cessation of NA in HBeAg-negative patients (Clinical trials number NCT ). All patients gave informed consent to participate in the study.

4 Patients were enrolled from 38 centres in 7 countries in the Asian-Pacific region. Inclusion criteria were age between years and treatment with NA for at least 2 years. Included patients were HBeAg-negative and anti-hbe Abs positive for at least 1 year prior to screening. Of note, the majority of patients (85.9%) were HBeAg negative prior to introduction of NA therapy. HBV DNA were < 40 IU/mL and ALT and AST levels upper limit of normal for at least 1 year prior to screening and at screening. Exclusion criteria were a platelet count < 100,000/mm 3 or platelet count < 150,000/mm 3 with esophageal varices on imaging and spleen size > 12 cm, or liver stiffness of 11 kilopascal [kpa] as measured by elastography using FibroScan or an AST to Platelet Ratio Index > 2, hepatocellular carcinoma, decompensation, co-infection, concomitant liver disease, immunosuppressive disorder/treatment or treatment with PEG-IFN in the last 12 months prior to screening. After 24 weeks, NA therapy was stopped in all patients. Follow-up was until 24 weeks or NA retreatment. Patients were followed every 2 weeks during the first 8 weeks, and then every 4 weeks. The primary end-point of the study was virological remission (HBV-DNA levels <40 IU/ml) until 24 weeks after stopping NA. Importantly, ABX203 did not influence the virological relapse, when administered simultaneously with NA therapy, as shown recently (8). All 224 patients treated with entecavir or tenofovir were selected (figure S1). 4 patients were subsequently excluded for missing follow-up data. Laboratory tests in this study were determined by standard procedures. Virological relapse was defined as HBV DNA > 200 IU/ml or 2,000 IU/ml, the test was not repeated in case of virological relapse. The COBAS AmpliPrep/COBAS TaqMan HBV Test, version 2.0 assay with a lower limit of quantification of HBV DNA <20IU/ml was used. Graph Pad Prism 6.0 (GraphPad Software, Inc, La Jolla, CA, USA) and SPSS 24.0 (IBM, Ehningen, Germany) were used for statistical analysis. All data are presented either as absolute numbers or as mean ± range unless otherwise stated. Continuous data were analyzed with Mann-Whitney test and categorical data with Fisher s exact test. Log-rank test was used for the Kaplan-

5 Meier survival analysis. Multiple logistic regression was used to identify parameters associated with virological relapse between baseline and week 12 and week 16 and 24. Results: In total, 220 patients primarily from Asia treated with entecavir (n=154) or tenofovir disoproxil fumarate (n=66) were included in the present study (table 1). Overall, virological relapse defined by HBV DNA > 2,000 IU/ml was documented in 54/220 (24.5%) patients until week 12 and in 126/220 (57.3%) patients until week 24. Thus, late relapse between week 16 and 24 occurred in 72 (32.7%) patients. Among the patients treated with tenofovir, 71% (47/66) had virological relapse until week 12, whereas 4.5% of patients treated with entecavir had virological relapse (7/154) in the same period (p<0.0001). Type of antiviral therapy, gender and age was significantly different between patients with early versus late relapse (table 1). To assess factors associated with early vs. late relapse, we performed a multiple logistic regression for antiviral therapy, gender, age, ALT, HBeAg status prior to NA therapy and HBsAg for early (week 0-12) vs late (week 16-24) virological relapse, defined as HBV DNA >2,000 IU/ml. Whereas no significant differences were detected for ALT, gender, age, HBeAg status and level of HBsAg, only the type of antiviral (entecavir versus tenofovir) was significantly associated with the timing of virological relapse (p< 0,001, table 1). The median time to virological relapse >2,000 IU/ml HBV DNA was 6 weeks after tenofovir discontinuation and 24 weeks after entecavir discontinuation (p < 0,0001, figure 1). This was similar if virological relapse was defined more restrictively as HBV DNA increase >200 IU/ml (figure S2). An additional analysis of only HBeAg negative patients prior to NA initiation (n=189) shows a similar result (figure S3).

6 Discussion: Our results show that the type of NA treatment affects the timing of HBV relapse after cessation of therapy. We here validate recent findings by Su et al., that virological relapse occurs much later after cessation of entecavir compared with tenofovir (9). This finding has important implications. Until now, no major difference between entecavir and tenofovir in terms of antiviral efficacy or patients outcomes are known for treatment naïve patients and the choice of treatment was up to the physician and mainly based on lamivudine resistance, costs or certain comorbidities (i.e. renal insufficiency) (10,1,2). Given the present findings, there may be more to the choice of NA. First, patients treated with entecavir will require a close follow-up even more than 3 months after treatment discontinuation. Second, and maybe even more importantly, the timing of HBV DNA relapse after treatment cessation might influence the impact on HBsAg decline, e.g. by different timing of inducing immune responses after stopping therapy. Similar findings were observed in our recently published small pilot trial in mainly European patients with a follow-up of 48 weeks (5). All patients who stopped entecavir experienced a late HBV DNA relapse after 12 weeks and all patients who stopped tenofovir experienced a relapse until week 8 (Figure S4, not shown previously (5)). Interestingly, only patients with an early relapse had an HBsAg decline >1 log at week 48 in our previous study (5). The reason for the difference between entecavir and tenofovir is unknown. Potential reasons are differences in pharmakokinetics or interaction with immune responses. Although half-life of the active compound of tenofovir and entecavir is similar, terminal elimination of entecavir may take up to 149 hours (11), however, this hardly explains the difference of several weeks to virological relapse we observed here. Recently, it was described that tenofovir, but not entecavir, leads to an induction of interferon (IFN)-λ (12). Indeed, it is not clear if tenofovir or entecavir is special compared to all other nucleos(t)ide analogues. A retrospective study has shown that the relapse after stopping lamivudine or telbivudine therapy was also faster than the relapse after entecavir treatment (13). Future

7 studies are most warranted to unveil the mechanistic process involved in entecavir prolonged HBV DNA suppression. Our study has some limitations. Given the follow-up of 24 weeks it is likely that some patients treated with entecavir will suffer from virological relapse at a later point in time, but, relapse at a later point in time would not change our conclusion. However, a bias cannot fully be excluded as some contributing factors as duration of NA therapy > 2 years or presence of NA resistance were not assessed and virological relapse was not confirmed immediately by a repeated test. In Asia, entecavir was approved earlier than tenofovir, however, this was not an issue for our European cohort, which showed similar results (5). However, time of consolidation therapy may influence the timing of relapse. Due to the short follow-up (until relapse or week 24) we could not investigate if the timing of relapse affects HBsAg decline or HBsAg loss rates. In addition, the study was not designed to address different dynamics of virological relapse between tenofovir and entecavir. Nevertheless, the study was prospective and the primary endpoint was virological remission, paralleling a study design that would be suitable to assess differences between tenofovir and entecavir. Our study as well as the study by Su et al. was conducted in Asian patients. Whether it will also be true for non- Asian patients remain to be confirmed, although our small study showed similar patterns in mainly Caucasian patients (5). In comparison to the study by Su et al., our study included more than twice as many patients and more than 85% of patients were HBeAg negative before introduction of NA therapy. In summary, this is the largest prospective study showing that the type of nucelos(t)ide analogue affects the relapse after cessation of therapy. This is an important finding for the clinical practice as well as for future studies addressing whether treatment discontinuation has an impact on functional cure of HBV infection. Conflict of interest: CHZS has received travel grants from Novartis and Gilead. AJH has nothing to disclose.

8 WT has nothing to disclose. PT has nothing to disclose. WWS has nothing to disclose. GEGN has patents protecting the ABX203 (NASVAC) vaccine. PG has nothing to disclose. JN has nothing to disclose. SC has nothing to disclose. SS has nothing to disclose. MPM reports grants and personal fees from Roche, grants and personal fees from Bristol Myers Squibb, grant and personal fees from Gilead, grants and personal fees from Novartis, grants and personal fees from GlaxoSmithKline, grants and personal fees from Medgenics, grants and personal fees from Enyo Pharma and grants and personal fees from Curevac outside of the submitted work. CT has nothing to disclose. HW reports grants, personal fees and non-financial support from Abbott, grants, personal fees and non-financial support from Roche Diagnostics, personal fees from Siemens, grants and personal fees from BMS, grants and personal fees from Gilead, grants and personal fees from Novartis, grants and personal fees from Roche, personal fees from Janssen, grants and personal fees from Merck/MSD, grants and personal fees from Abbvie, grants and personal fees from Eiger, grants and personal fees from Falk and Falk Foundation, other from Transgene, non-financial support and other from MyrmbH, outside the submitted work. MC has received fees for consulting lectures and / or Board membership from Roche, MSD, Gilead, BMS, Janssen, Abbvie and Roche, outside of the submitted work.

9 Funding: The study was funded by ABIVAX, Paris, France. Author contributions: CHZS, HW and MC: study concept and design, analysis and interpretation of data, drafting of the manuscript; critical revision of the manuscript, statistical analysis, study supervision MPM drafting of the manuscript, critical revision of the manuscript, technical and material support; study supervision AJH, WS, PT, WWS, GEGN, PG, JN, SC, SS, CT, acquisition of data, critical revision of the manuscript, technical and material support References 1. Sarin SK, Kumar M, Lau GK, et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. Hepatol Int. 2016; 10(1): Lampertico P, Agarwal K, Berg T, et al. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. 2017; 67(2): Papatheodoridis G, Vlachogiannakos I, Cholongitas E, et al. Discontinuation of oral antivirals in chronic hepatitis B: A systematic review. Hepatology. 2016; 63(5): Berg T, Simon KG, Mauss S, et al. Long-term response after stopping tenofovir disoproxil fumarate in non-cirrhotic HBeAg-negative patients - FINITE study. J Hepatol. 2017; 67(5): Höner zu Siederdissen C, Rinker F, Maasoumy B, et al. Viral and Host Responses After Stopping Long-term Nucleos(t)ide Analogue Therapy in HBeAg-Negative Chronic Hepatitis B. J Infect Dis. 2016; 214(10): Jeng W-J, Chen Y-C, Chien R-N, Sheen I-S, Liaw Y-F. Incidence and predictors of

10 HBsAg seroclearance after cessation of nucleos(t)ide analogue therapy in HBeAg negative chronic hepatitis B. Hepatology. 2017; : /hep Zimmer CL, Rinker F, Höner zu Siederdissen C, et al. Increased NK Cell Function After Cessation of Long-Term Nucleos(t)ide Analogue Treatment in Chronic Hepatitis B Is Associated With Liver Damage and HBsAg Loss. J Infect Dis. 2018;. EPUB 8. Wedemeyer H, Hui AJ, Sukeepaisarnjaroen W, et al. Therapeutic vaccination of chronic hepatitis B patients with ABX203 (NASVAC) to prevent relapse after stopping NUCs: contrasting timing rebound between tenofovir and entecavir. J Hepatol. Elsevier; 2017; 66(1):S Su T-H, Yang H-C, Tseng T-C, et al. Distinct relapse rates and risk predictors after discontinuing tenofovir and entecavir therapy. J Infect Dis. 2018; : /infdis/jix Terrault NA, Bzowej NH, Chang K-M, Hwang JP, Jonas MM, Murad MH. AASLD guidelines for treatment of chronic hepatitis B. Hepatology. 2016; 63(1): Yan J-H, Bifano M, Olsen S, et al. Entecavir Pharmacokinetics, Safety, and Tolerability After Multiple Ascending Doses in Healthy Subjects. J Clin Pharmacol. Blackwell Publishing Ltd; 2006; 46(11): Murata K, Asano M, Matsumoto A, et al. Induction of IFN-λ3 as an additional effect of nucleotide, not nucleoside, analogues: a new potential target for HBV infection. Gut. 2016; :gutjnl Jeng W-J, Sheen I-S, Chen Y-C, et al. Off-therapy durability of response to entecavir therapy in hepatitis B e antigen-negative chronic hepatitis B patients. Hepatology. 2013; 58(6):

11 Parameter Screening (24 weeks before BL) Baseline (NA discontinuation) Early Relapse (week 0-12) Late Relapse (week 16-24) P (early vs late relapse) No of patients 220 (100%) 54 (24.5%) 72 (32.7%) Female 65 (29,5%) 8 (15%) 29 (40%) Male 155 (70,5%) 46 (85%) 43 (60%) Antiviral therapy Entecavir 154 (70%) 7 (13%) 68 (94%) < Tenofovir 66 (30%) 47 (83%) 4 (6%) Genotype (n = 34) A 2 (6%) 0 2 (7%) B 19 (56%) 3 (43%) 16 (59%) (GT b vs GT c) C 13 (38%) 4 (57%) 9 (33%) D Vaccine (ABX-203) + NA 149 (67.7%) 37 (68.5%) 49 (68.1%) 1.0 HBeAg negative at NA introduction 189 (85.9%) 51 (94.4%) 65 (90.3%) Fibroscan (kpa)* 5.2 ( ) 5 ( ) 5.3 ( ) Age (years) 52 (20 66) 50 (26-65) 56 (39-64) Albumin (g/dl) 46 (39-54) 46 (38-54) 46 (41-54) 46 (42-50) ALT (U/l) 20 (6 55) 21 (9-54) 22 (9-51) 21 (12 40) Bilirubin (mg/dl) 9,5 (3 36) 9 (0-32) 8 (0-32) 9 (4-28) 0.56 HBsAg (U/l) 801 ( ) 712 ( ) 632 ( ) 659 ( ) Hemoglobin (g/l) 148 ( ) 150 ( ) 150 ( ) 144 ( ) Platelets (/nl) 186 (81 432) 210 (93-523) 202 ( ) 219 ( ) Pts with platelets <150/nl 39 (17.7%) 19 (8.3%) 8 (14.8%) 4 (5.5%) Multiple logistic regression of early virological relapse (week 0 12) vs late virological relapse (week 16-24) Sig. Odds ratio Confidence interval Antiviral therapy Gender Age ALT (U/l) HBsAg (U/l) HBeAg status Table 1: Baseline criteria at the timepoint of nucleos(t)ide analogue discontinuation and multiple logistic regression analysis for virological relapse (HBV DNA > 2,000 IU/ml) week 0-12 vs week Continuous data presented as median + range; baseline: time point of NA discontinuation; screening: time point of screening for the vaccine study (24 weeks before NA discontinuation). *data available for 150 patients. Data on patients without relapse are shown in the supplemental table.

12 Figure 1.

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