Journal Pre-proof

Safety and effectiveness of up to 3 years’ bulevirtide monotherapy in patients withHDV-related cirrhosis

Alessandro Loglio, Peter Ferenci, Sara Colonia Uceda Renteria, Christine Y.L.Tham, Caroline Scholtes, Heidemarie Holzmann, Florian van Bömmel, Marta Borghi,Riccardo Perbellini, Alessandro Rimondi, Elisa Farina, Elena Trombetta, MariaManunta, Laura Porretti, Daniele Prati, Ferruccio Ceriotti, Fabien Zoulim, AntonioBertoletti, Pietro Lampertico

PII: S0168-8278(21)02119-X

DOI: https://doi.org/10.1016/j.jhep.2021.10.012

Reference: JHEPAT 8478

To appear in: Journal of Hepatology

Received Date: 17 June 2021

Revised Date: 27 September 2021

Accepted Date: 12 October 2021

Please cite this article as: Loglio A, Ferenci P, Uceda Renteria SC, Tham CYL, Scholtes C, HolzmannH, van Bömmel F, Borghi M, Perbellini R, Rimondi A, Farina E, Trombetta E, Manunta M, PorrettiL, Prati D, Ceriotti F, Zoulim F, Bertoletti A, Lampertico P, Safety and effectiveness of up to 3 years’bulevirtide monotherapy in patients with HDV-related cirrhosis, Journal of Hepatology (2021), doi: https://doi.org/10.1016/j.jhep.2021.10.012.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the additionof a cover page and metadata, and formatting for readability, but it is not yet the definitive version ofrecord. This version will undergo additional copyediting, typesetting and review before it is publishedin its final form, but we are providing this version to give early visibility of the article. Please note that,during the production process, errors may be discovered which could affect the content, and all legaldisclaimers that apply to the journal pertain.

© 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

1

Safety and effectiveness of up to 3 years’ bulevirtide monotherapy in patients with HDV-1

related cirrhosis 2

3

Alessandro Loglio1, Peter Ferenci

2, Sara Colonia Uceda Renteria

3, Christine Y.L. Tham

4, Caroline 4

Scholtes5, Heidemarie Holzmann

6, Florian van Bömmel

7, Marta Borghi

1, Riccardo Perbellini

1, 5

Alessandro Rimondi8, Elisa Farina

8, Elena Trombetta

9, Maria Manunta

10, Laura Porretti

9, Daniele 6

Prati10

, Ferruccio Ceriotti3, Fabien Zoulim

5, Antonio Bertoletti

4, Pietro Lampertico

1,8 7

8

1) Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Division of Gastroenterology 9

and Hepatology, Milan, Italy; 2) Department of Internal Medicine III, Division of 10

Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria; 3) Foundation 11

IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Virology Unit, Milan, Italy; 4) Program 12

Emerging Infectious Diseases, Duke-NUS Medical School, Singapore; 5) Hospices Civils de 13

Lyon, INSERM Unit 1052, Lyon University, France; 6) Center for Virology, Medical University 14

of Vienna, Vienna, Austria; 7) Section of Hepatology, Department of Gastroenterology, 15

University Hospital Leipzig, Leipzig; 8) CRC “A. M. and A. Migliavacca” Center for Liver 16

Disease, Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy; 17

9) Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Flow cytometry service, Milan, 18

Italy; 10) Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Department of 19

Transfusion Medicine and Hematology, Biobank POLI-MI, Milan, Italy 20

21

Corresponding Author: 22

Journ

al Pre-

proof

2

Pietro Lampertico, MD, PhD 1

CRC “A. M. and A. Migliavacca” Center for Liver Disease Division of Gastroenterology and 2

Hepatology 3

Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico University of Milan 4

Via F. Sforza 35 - 20122 Milan, Italy Tel: +39-0255035432 5

Fax: +39-0250320410 6

e-mail: pietro.lampertico@unimi.it 7

8

Keywords: 9

Bulevirtide; HDV; Entry inhibitor; T-cell; HDV-RNA; HBcrAg; HBV-RNA; HBV 10

11

Main text word count: 2581 12

Abstract word count: 256 13

No. of tables: 1 14

No. of figures: 1 15

No. of supplementary tables: 1 16

17

Financial Supports: 18

This work was supported by a grant from “Ricerca Corrente RC2021/105-01”, Italian Ministry of 19

Health, and by a grant from the French National Research Agency Investissements d’Avenir 20

Progam (CirB-RNA project-ANR-17-RHUS-0003)21

Journ

al Pre-

proof

3

1

Conflict of interest: 2

Alessandro Loglio: travel grant for MYR Pharma, speaker bureau for Gilead Sciences. Peter 3

Ferenci: advisor and speaker bureau for Gilead Sciences, GSK, MSD, Abbvie; Florian van 4

Bömmel: research grants from Gilead Sciences Inc., MYR Pharma and Roche Diagnostics, 5

speaker and advisor for Gilead Sciences, Roche, Janssen, Abbvie, MSD and BMS; Antonio 6

Bertoletti advisor for Gilead, Spring-Bank, Vir, Simcere; he is also Scientific Founder of LION 7

TCR pte.; Fabien Zoulim: advisor for Aligos, Antios, Arbutus, Assembly, Gilead, GSK, MYR 8

Pharma, Roche; Pietro Lampertico: advisor and speaker bureau for BMS, Roche, Gilead Sciences, 9

GSK, MSD, Abbvie, Janssen, Arrowhead, Alnylam, Eiger, MYR Pharma, Antios, Aligos. The 10

other authors declare that they have no competing interests. 11

12

Authors’ contributions: 13

AL, PF and PL were involved in patients’ care and drafting of the manuscript. AL, HH, MB, AR, 14

EF, RP were involved in data collection. ET, MM, LP, DP were involved in PBMC extraction and 15

cryopreservation. CT and AB performed T-cell analysis. CS, FvB and FZ performed HBV-RNA 16

and HBcrAg analysis. HH, SU, FC performed molecular and virological analysis. AL, AB, PF, FZ 17

and PL were involved in manuscript editing. All authors approved the final version of the 18

manuscript. 19

20

Data availability statement 21

The data is available after a justified request. 22

Journ

al Pre-

proof

4

ABSTRACT 1

The entry-inhibitor Bulevirtide (BLV) received conditional approval by EMA in July 2020 for 2

the treatment of adult patients with compensated chronic hepatitis Delta. However, the 3

effectiveness and safety of BLV administered as monotherapy beyond 48 weeks in difficult to 4

treat HDV cirrhotic patients is presently unknown. Here we describe the first patients with HDV-5

related compensated cirrhosis who were treated with BLV (10 mg/day as a starting dose) for up to 6

three years as compassionate use. Patients were also monitored for HBcrAg and HBV-RNA levels 7

and HDV and HBV specific T-cells markers. 8

In the patient who stopped BLV at week 48 after achieving a virological and biochemical 9

response, the initial virological and biochemical rebound was followed by ALT normalization 10

coupled with low HDV-RNA and HBsAg levels. In the two patients treated continuously for 3 11

years, virological and biochemical responses were maintained throughout the treatment period 12

even after dose reduction. In a patient with advanced compensated cirrhosis, liver function tests 13

significantly improved, esophageal varices disappeared, and histological/lab features of 14

autoimmune hepatitis resolved. Overall, no safety issues were recorded, as bile salt increase was 15

asymptomatic. While serum HBV-RNA levels remained undetectable in all patients, HBcrAg 16

levels showed a progressive, yet modest decline during long-term BLV-treatment. No HDV-17

specific Interferon-γ producing T-cells were detected, neither after HDV reactivation (after BLV 18

withdrawn in Patient 1) nor during 3 years of BLV treatment. In conclusion, this report shows that 19

continuous administration of BLV monotherapy for three years provides excellent virological and 20

clinical response in HDV cirrhotic patients who had contraindications to IFN-based therapies.21

Journ

al Pre-

proof

5

LAY SUMMARY 1

- HDV-RNA levels became undetectable, and ALT normalized in all three patients treated with 2

Bulevirtide (BLV). Virological and biochemical responses were maintained even after dose 3

reduction. 4

- Improvement of liver function tests, regression of esophageal varices and recovery of HDV-5

related autoimmune disease were documented in the male cirrhotic patient long-term treated 6

with BLV. 7

- An asymptomatic increase of bile acids was the only drug-related clinical adverse event. 8

Journ

al Pre-

proof

6

INTRODUCTION 1

Chronic hepatitis Delta (CHD) is a rare but severe form of chronic viral hepatitis that affects 2

approximately 12-72 million patients worldwide[1]. It is sustained by the hepatitis D virus 3

(HDV), a small defective virus that requires the helper function of HBV to replicate and 4

propagate[2]. In the last 30 years, the only therapeutic approach has been the off-label use of a 48-5

week course of Interferon (IFNα)[3]. However, this antiviral strategy is characterized by limited 6

off-therapy responses and an unfavorable safety profile[3,4]. 7

The unmet medical need of an effective and safe therapeutic option for CHD patients coupled 8

with the recent identification of the entry receptor for HBV and HDV, has fostered the research 9

in this field. Among the several compounds now being tested in clinical studies, Bulevirtide 10

(BLV) is the only drug that has received a conditional approval from the European Medicine 11

Agency in July 2020, at the dose of 2 mg/day s.c.[4]. Previously named Myrcludex–B and now 12

commercialized as Hepcludex® in European Union, BLV is a first in class entry-inhibitor of HBV, 13

a subcutaneously delivered lipopeptide that mimics the Na+-taurocholate co-transporting 14

polypeptide (NTCP) receptor binding domain, blocking the HDV/HBV entry exclusively in liver 15

cells. In Phase II trials, BLV administration induced HDV-RNA reduction and ALT improvement 16

during a 24- and 48-week treatment that was however followed by a relapse after treatment 17

cessation in most patients. When combined with Peg-IFNα, a synergic effect on HDV-RNA and 18

HBsAg decline was demonstrated[5]. 19

In this brief report we describe for the first time the safety, effectiveness and clinical response to 20

BLV administered for up to 3 years as a monotherapy in patients with HDV-related compensated 21

cirrhosis. In addition, we enriched the study by an integrated analysis of both standard and 22

Journ

al Pre-

proof

7

innovative HBV markers, such as serum HBV core-related Antigen (HBcrAg) and HBV-RNA, 1

coupled with an immunological monitoring of IFN-γ HDV and HBV specific T-cells. 2

3

PATIENTS AND METHODS 4

This brief report study is based on the data generated in three CHD patients who received BLV in a 5

single patient compassionate use program. All instances of compassionate use were approved by a 6

local Ethic Committee [6] and informed consent was obtained for all subjects, according to 7

Helsinki Declaration. BLV at 10 mg as the initial dose was self-administered as 8

subcutaneous injections every 24 hours. Liver function tests, total bile acids and virological HDV 9

and HBV markers were monitored every 4 weeks for 2 years and then every 8-weeks; liver 10

stiffness by Fibroscan® every 6 months, upper endoscopy according to Baveno VI 11

recommendations, and HCC surveillance as per international guidelines[3]. 12

HDV-RNA was quantified by RoboGene® (HDV-RNA quantification 2.0; Aj-Roboscreen, Jena, 13

Germany; lower limit of detection (LOD) 6 IU/mL); HBV-RNA was quantified by an in-house 14

real-time PCR technique (Leipzig, LOD 160 cp/mL) in the first year, and by a real-time PCR 15

based investigation assay (Roche Diagnostics, Pleasanton, Ca, USA, LLOQ 10 cp/mL) in the 16

following 2 years[7]. Serum HBcrAg levels were measured using LUMIPULSE® G HBcrAg 17

assay (Fujirebio Europe, LOD 2 log10 U/mL). HBV DNA was quantified by Abbott RealTime 18

HBV (Abbott Diagnostics, Rome, Italy; LOQ 10 IU/mL) or by Roche Cobas® 19

(AmpliPrep/TaqMan System®, LOQ 20 IU/mL). HBV genotype was determined by INNO-LiPA 20

HBV genotyping (Fujirebio Europe NV, Ghent, Belgium), while HDV genotype was assessed by 21

sanger sequencing and analyses of the hepatitis delta antigen region. HDV RNA was transcribed 22

using primers random hexamers and SuperScript III First-Strand Synthesis System for RT-PCR 23

Kit (Invitrogen, California, USA). First PCR and Nested PCR were performed using primers 24

Journ

al Pre-

proof

8

synthesized by metabion international AG (Metabion GmbH, Germany) and TaKaRa Ex Taq Hot 1

Start Version Kit (TAKARA BIO INC, Kusatsu, Japan). In peripheral blood mononuclear cells 2

(PBMC) collected and cryopreserved every 4 weeks, HDV/HBV-specific T-cell quantity and 3

function were analyzed by direct ex-vivo IFN-γ enzyme-linked immunosorbent spot (ELISPOT) 4

assays, using a panel of 313 overlapping peptides (15-mers overlapping by 10AA) covering the 5

full proteome sequence of HBV genotype D (Accession number AF121241) pooled in 8 6

individual mixtures containing the indicated peptides number: HBV-X (29 peptides), 7

Nucleocapsid (41 peptides), envelope 1 and 2 (38-peptides each), polymerase 1, 2, 3, and 4 8

(42,42,42 and 41 peptides, respectively)[8]. Another panel of 42 overlapping peptides (15-mers 9

overlapping by 10 AA) covers the full proteome sequence of HDV genotype 1. Both HBV- and 10

HDV-specific T-cell responses were analyzed in IFN-γ ELISPOT assays ex vivo. Briefly, 96-well 11

plates (Multiscreen-HTS; Millipore, Billerica, MA) were coated overnight at 4°C with 5 μg/ml 12

capture mouse anti-human IFN-γ monoclonal antibody (Purified NA/LE anti-human IFN-γ). 13

Plates were then blocked with RPMI medium 1640 containing 10% Fetal Bovine Serum (FBS) 14

and 1% Penicillin-Streptomycin–L-Glutamine for 2 hours. A total of 3×105 PBMCs were seeded 15

per well for each individual peptide mixture. Plates were incubated for 18 hours at 37°C in the 16

absence or presence of peptides (at a final concentration of 2 μg/ml). After the incubation, plates 17

were incubated with biotinylated anti-human IFN-γ and streptavidin-HRP and developed using 18

the chromogen substrate solution according to the recommended protocol from BD (Becton 19

Dickinson). The colorimetric reaction was stopped after 10 to 15 minutes by washing the plates 20

with distilled water. Plates were air dried, and spots were counted using an automated ELISPOT 21

reader (ImmunoSpot reader; CTL Technologies, OH). The number of peptide-specific IFN-γ-22

secreting cells was calculated by subtracting the non-stimulated control value from that of the 23

stimulated sample. Positive controls consisted of PBMCs stimulated with phorbol myristate 24

Journ

al Pre-

proof

9

acetate (10 ng/ml) and ionomycin (100 ng/ml). In the direct ex vivo assays, wells were considered 1

positive when the number of spot-forming units (SFU) was above 5 and at least three times the 2

mean value of the unstimulated control wells. Negative control was stimulated with RPMI 3

containing the DMSO concentration present in the diluted peptide mixtures (0.2%). Only 4

experiments with a positive result in the positive control were considered[6,9]. 5

The first 48-week results of these three patients have been previously published[6]. All patients 6

were already under Tenofovir Disoproxil Fumarate (TDF) treatment. 7

8

RESULTS 9

Patient 1 10

A 69 year-old, Caucasian, HBeAg-negative female with HDV-related compensated cirrhosis 11

complicated by portal hypertension (splenomegaly and thrombocytopenia) that contraindicated 12

IFN therapy. Comorbidities included diabetes mellitus, femoral and lumbar osteopenia, mild 13

arterial hypertension and uterine polyps under assessment. This patient had genotype D of HBV 14

and genotype 1 of HDV; she was treated with TDF. 15

During 10 mg/day BLV treatment, ALT rapidly normalized, HDV-RNA became undetectable and 16

clinical parameters improved (Figure-Panel A, Supplementary Table). Following the diagnosis of 17

an endometrial carcinoma at week 52, BLV was withdrawn as a precautious measure to avoid 18

any possible drug-to-drug interaction with chemotherapy. After BLV discontinuation, HDV-19

RNA rapidly rebounded and ALT flared up peaking at week 28 (333 IU/L), but both markers 20

fell within the normal range by week 48 off-therapy (Figure-Panel A, Supplementary Table). 21

HDV reactivation was not associated to any sign of clinical decompensation. HBsAg levels, that 22

had slightly increased during BLV treatment, progressively and significantly declined after BLV 23

was withdrawn, paralleling HDV-RNA rebound and ALT flare (Figure-Panel A). In summary, at 24

Journ

al Pre-

proof

10

96-week off-therapy, HBsAg and HDV-RNA levels were very low and ALT normal. No 1

significant increases in HDV or HBV specific IFN-γ T-cell response was observed neither during 2

BLV treatment nor during virological and clinical relapse, nor HBcrAg levels that remained 3

always below the LOD except at BLV baseline and at ALT peak secondary to HDV relapse 4

(Figure-Panel A). 5

Bile acids, that increased during BLV administration, rapidly normalized after drug withdrawal. 6

The asymptomatic increase of serum bile acids levels >160 µmol/l that we observed at week 20 7

and 24 was likely due to the fact that BLV was self-administered before blood sampling, and not 8

after as usually done. 9

10

Patient 2 11

The second patient was a 51 year-old male, Caucasian, HBeAg-negative with HDV-compensated 12

cirrhosis complicated by thrombocytopenia, small esophageal varices (EV) and autoimmune 13

hepatitis (liver biopsy performed in 2010) that contraindicated IFN-treatment[6]. He had diabetes 14

on diet therapy (Table). This patient, on TDF therapy, had genotype D of HBV and genotype 1 of 15

HDV. 16

Treatment with BLV 10 mg/day induced a rapid biochemical and virological responses that was 17

maintained up to week 144 without any sign of virological or biochemical breakthrough even 18

after BLV dose reduction to 5 mg/day at week 76 (Figure-Panel B): we decided to reduce BLV 19

daily dose only to simplify therapy, i.e. only one injection every morning, given the excellent 20

HDV suppression lasting for >1 year. During BLV treatment, liver function tests as well as alpha-21

fetoprotein (AFP) and IgG levels rapidly normalized, platelet count improved to almost normal 22

levels, while HBsAg levels and the other HBV markers remained stable over time. Serum HBV-23

RNA levels remained undetectable while HBcrAg levels showed a progressive, yet modest 24

Journ

al Pre-

proof

11

decline (from 4.5 to 3.4 Log U/mL). In a liver biopsy performed after 72 weeks of BLV 1

treatment, we observed a reduction of plasma cells infiltration compared to what had been 2

observed in the previous liver biopsy performed in 2010, with an improvement of histological 3

features of autoimmune hepatitis. 4

Notably, platelet count significantly increased, liver stiffness improved, and esophageal varices 5

disappeared after 20 months of BLV treatment (Table). The latter finding was confirmed in two 6

consecutive endoscopies 12-months apart. No recovery of HDV nor HBV specific IFN-γ 7

producing T-cells function was observed at any time point during BLV administration (Figure-8

Panel B). BLV was well-tolerated as no local or systemic adverse events were reported, the 9

increase of bile acids levels was dose-related and fully asymptomatic. 10

11

Patient 3 12

The third patient was a 58 year-old female, native to Uzbekistan, HBeAg-negative with HDV-13

related compensated cirrhosis complicated by an autoimmune thrombocytopenia with detectable 14

anti-platelet antibodies for which she was treated in the past with high doses of steroids (Table). 15

This patient, on anti-HBV therapy with TDF, had genotype D of HBV and genotype 1 of HDV. 16

BLV administration led to a rapid biochemical response that was followed by a virological 17

response with HDV-RNA becoming undetectable at week 52, both responses were maintained up 18

to week 144 despite BLV dose reduction to 5 and 2 mg/day (Figure-Panel C). Liver function tests 19

as well as HBV markers remained stable over time while HBcrAg levels showed a progressive but 20

modest decline (Table, Figure-Panel C). No local or systemic side effects were recorded apart 21

from a dose-related, fully asymptomatic increase of bile acid, that had a decrease from a mean 22

level of 203 to 152 umol/L after BLV dose reduction (105 mg/day). Autoimmune 23

thrombocytopenia did not recur. 24

Journ

al Pre-

proof

12

1

DISCUSSION 2

To our knowledge this is the first report assessing the safety, effectiveness and clinical response 3

of BLV administered as monotherapy for up to three years in patients with HDV-related 4

compensated cirrhosis in whom Peg-IFN therapy was contraindicated. The long-term 5

administration of BLV monotherapy in these difficult to treat patients was associated with 6

positive virological and clinical results coupled with a favorable safety profile. All patients 7

reported excellent compliance to BLV therapy (2 subcutaneous injections every morning), as 8

confirmed by the increase of bile salts levels, a biomarker of target engagement. 9

One of the most important findings of the study is the positive virological and biochemical 10

response observed as all three patients achieved and maintained undetectable HDV-RNA, which 11

was tested by a very sensitive assay. These findings are unprecedented for at least three reasons: 12

first, no studies to date have reported the outcome of BLV treatment beyond week 48; second, no 13

data exists to demonstrate that virological response was maintained upon dose reduction; third, 14

the effectiveness of the administration of this drug for such a long-term in difficult to treat 15

patients such as those with advanced compensated cirrhosis, including one case with clinical 16

significant portal hypertension (CSPH), has never been reported so far. 17

Another important finding was the clinical response that we have observed in these patients. One 18

patient who had compensated cirrhosis with CSPH, showed a clinically relevant improvement of 19

liver function tests, AFP and platelet levels, as well as portal hypertension features, with the 20

regression of EV. To our knowledge, this is the first report of EV disappearance in a HDV patient, 21

an event that has been already demonstrated in patients with HBV-related cirrhosis long-term 22

treated with nucleos(t)ide analogs[3]. In the other two cirrhotic patients, liver function tests and 23

synthetic function of the liver remained stable up to 3 years of therapy, a favorable finding for a 24

Journ

al Pre-

proof

13

chronic liver disease characterized by high rates of progression to end-stage-liver disease and 1

HCC[2]. 2

There is currently no safety data published or presented beyond week 48 with BLV in HDV 3

patients, regardless of the severity of liver disease. Extension of BLV treatment to 3 years in these 4

two compensated HDV cirrhotic patients was well tolerated, not associated with any drug-related 5

local or systemic adverse event, confirming the preliminary favorable week 48 data from phase II 6

studies[4,5]. No itching was reported even though high levels of bile acids were observed in 7

patients with advanced liver disease treated with BLV 10 mg/day, first results of an observation 8

lasting 3 years in a real-life setting. 9

It is well known that a significant proportion of the patients with viral hepatitis may have or may 10

develop autoimmune phenomena via molecular mimicry, ranging from isolated non-organ 11

specific autoantibodies positivity to florid autoimmune hepatitis. Patient 2 was indeed a 12

representative case, with hypergammaglobulinemia, autoantibodies and interface hepatitis with 13

plasma cells infiltration, associated with CHD. Upon achieving virological response to BLV, the 14

laboratory and histological features of autoimmunity rapidly improved, a strong argument in favor 15

of a causal relationship between HDV replication and autoimmunity. As far as we know, this is 16

the first case of HDV-related autoimmune hepatitis cured by antiviral therapy. A similarly 17

favorable outcome was observed in Patient 3, who did not have a recurrence of autoimmune-18

mediated thrombocytopenia during BLV treatment. 19

These cases were also instrumental in assessing two recently developed HBV markers, such as 20

HBV-RNA and HBcrAg, in the setting of HDV patients treated with BLV[10]. In the two patients 21

treated with BLV for 3 years, moderate to high serum levels of HBcrAg were documented while 22

serum HBV-RNA was persistently undetectable. Since both markers are expected to mirror the 23

covalently close circular DNA (cccDNA) transcriptional activity of HBV, these findings may be 24

Journ

al Pre-

proof

14

related to the fact that HDV could directly interfere with HBV replication processes associated 1

with extremely low levels of cccDNA[4,11]. To confirm these results and investigate the 2

underlying molecular mechanisms, additional studies are needed. 3

Last but not least, we were able to perform immunological studies in two BLV-treated patients. 4

No recovery of HBV or HDV-specific IFN-γ producing T-cell was identified ex vivo, regardless 5

of the duration of therapy. Of note, recovery of the peripheral HBV and HDV T-cell was not 6

observed even in Patient 1, despite a significant ALT flare following BLV discontinuation. Direct 7

ex-vivo Elispot can only detect robust expansion of HBV and HDV-specific T cell frequency and 8

we cannot exclude that more detailed analysis, such as T-cell analysis after a round of in-vitro 9

expansion[12] might be able to detect subtle modifications of frequencies. However, the lack of 10

recovery of circulating HBV and HDV-specific IFN-γ producing T-cells in these treated patients 11

could also be explained by the mature age of the patients (>50 years), and the preferential homing 12

of HBV and HDV specific T-cells into the liver[12]. 13

14

CONCLUSIONS 15

This is the first report that describes the safety, virological and clinical responses of BLV 16

monotherapy administered for up to 3 years in patients with HDV-related compensated cirrhosis 17

treated via compassionate use. Forty-years after the discovery of the Delta virus, the results 18

conveyed by this first report on long-term BLV monotherapy will pave the way for the 19

management of this severe form of chronic liver disease.20

Journ

al Pre-

proof

15

ABBREVIATIONS 1

HBcrAg, HBV core-related Antigen; CHD, chronic hepatitis Delta; cccDNA, covalently close 2

circular DNA; AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; EV, esophageal varices; 3

NUC, nucleos(t)ide analogs; TDF, tenofovir disoproxil fumarate; PBMC, peripheral blood 4

mononuclear cells; ALT, alanine aminotransferase; BLV, bulevirtide. 5

6

DECLARATIONS 7

The use of Bulevirtide was approved by the local Ethics Committee on a 6-month basis for the 8

Italian patients, with written informed consent from the Austrian patient. Informed consent for 9

additional analyses and serum storage was obtained by all patients. 10

11

ACKNOWLEDGEMENTS 12

We thank Dr. Alexander Alexandrov from MYR Pharma for the free supply of Bulevirtide, and 13

Fujirebio Italy for the free supply of HBcrAg kits. We thank Charlotte Fenwick, native English 14

speaker, for the language revision of the paper. 15

Journ

al Pre-

proof

16

REFERENCES 1

1) Stockdale AJ, Kreuels B, Henrion MYR, Giorgi E, Kyomuhangi I, de Martel C, et al. The 2

global prevalence of hepatitis D virus infection: Systematic review and meta-analysis. J 3

Hepatol. 2020;73:523–532 4

2) Rizzetto M, Hamid S, Negro F. The changing context of hepatits D. J Hepatol. 5

2021;74:1200-1211 6

3) European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on 7

the management of hepatitis B virus infection. J Hepatol 2017;67:370-98 8

4) Urban S, Neumann-Haefelin C, Lampertico P. Hepatitis D virus in 2021: virology, 9

immunology and new treatment approaches for a difficult-to-treat disease. Gut 2021;0:1–13. 10

doi:10.1136/gutjnl-2020-323888 11

5) Wedemeyer H, Schöneweis K, Pavel O, Chulanov V, Stepanova T, Viacheslav M, et al. 48 12

weeks of high dose (10 mg) bulevirtide as monotherapy or with peginterferon alfa-2a in 13

patients with chronic HBV/HDV coinfection. J Hepatol 2020;73:S52 14

6) Loglio A, Ferenci P, Uceda Renteria SC, Tham CYL, van Bömmel F, Borghi M, et al. 15

Excellent safety and effectiveness of high-dose Myrcludex-B monotherapy administered for 16

48 weeks in HDV-related compensated cirrhosis: A case report of 3 patients. J Hepatol. 17

2019;71:834-839 18

7) Scholtès C, Hamilton A, Scott B, , Wang L, Plissonnier ML, Berby F, et al. Performance of a 19

novel automated assay for the detection and quantification of HBV pregenomic 20

RNA/circulating RNAs in chronic HBV patients. Hepatology 2020;72(S1):447A 21

8) Tan AT, Loggi E, Boni C, Chia A, Gehring AJ, Sastryet KSR, et al. Host ethnicity and virus 22

genotype shape the hepatitis B virus-specific T-cell repertoire. J. Virol. 2008;82:10986–23

10997 24

Journ

al Pre-

proof

17

9) Tan AT, Koh S, Goh W, Zhe HY, Gehring AJ, Lim SG, et al. A longitudinal analysis of innate 1

and adaptive immune profile during hepatic flares in chronic hepatitis B. J Hepatol. 2

2010;52:330–339 3

10) Yurdaydin C, Abbas Z, Buti M, Cornberg M, Esteban R, Etzion O, et al. Treating chronic 4

hepatitis delta: The need for surrogate markers of treatment efficacy. J Hepatol. 5

2019;70:1008-1015 6

11) Tu T, Zehnder B, Qu B, Urban S. De novo synthesis of hepatitis B virus nucleocapsids is 7

dispensable for the maintenance and transcriptional regulation of cccDNA. JHEP Rep. 8

2020;3:100195 9

12) Le Bert N, Gill US, Hong M, Kunasegaran K, Tan DZM, Ahmad R, et al. Effects of hepatitis 10

B surface antigen on virus-specific and global T cells in patients with chronic hepatitis B 11

virus infection. Gastroenterology 2020;159:652–66412

Journ

al Pre-

proof

18

FIGURE LEGEND 1

2

Figure: Changes of HDV RNA, ALT, bile acids, HBsAg, HBcrAg, HBV DNA and HBV RNA 3

levels during Bulevirtide (BLV) treatment in the three patients. 4

(A) Patient 1 (BLV was administered just before blood sampling at week 20, 24, 28); 5

(B) Patient 2; (C) Patient 3; (D and E) Immunological parameters variation in Patient 1 6

and 2 (Bars show the numbers of spots x 105 PBMC responding to the different 7

peptides mixtures or PMA+Ionomicyne [Positive control] while in the horizontal axis 8

Time as weeks of treatment or off-treatment). 9

Journ

al Pre-

proof

19

1

Table – Time course of virological, biochemical, and clinical variables during BLV and TDF treatment in Patients 2 and 3 (reported on a 6-month basis). 2

3

Variables Patient 2 Patient 3

Baseline Week 24 Week 48 Week 72 Week 96 Week 120 Week 144 Baseline Week 24 Week 48 Week 72 Week 96 Week 120 Week 144

AST (U/L) / ALT (U/L) 179 / 232 38 / 25 38 / 24 34 / 23 37 / 26 35 / 23 32 / 20 111 / 244 42 / 46 33 / 35 37 / 35 37 / 29 25 / 30 29 / 22

ALP (U/L) / GGT (U/L) 185 / 231 89 / 138 86 / 140 84 / 128 66 / 103 74 / 107 72 / 77 74 / 44 95 / 19 87 / 17 92 / 14 86 / 13 85 / 15 91 / 14

Total bilirubin (mg/dL) 0.4 0.6 0.4 0.4 0.8 0.7 0.6 0.4 0.4 0.3 0.4 0.5 0.5 0.3

pCHE (U/L) 4,470 6,534 6,648 6,692 7,633 7,029 7,421 6,330 6,340 6,310 5,990 6,110 6,180 4,960

Albumin / Gamma globulin

(g/dL) 3.6 / 2.9 4.4 / 1.9 4.4 / 1.5 4.2 / 1.3 4.6 / 1.2 4.4 / 1.1 4.4 / 1.0 4.1 / - 4.5 / - 4.5 / - 4.6 / - 4.5 / - 4.6 / - 4.6 / -

IgG (mg/dL) 3,077 2,061 1,608 1,642 1,608 1,422 1,147 2,350 - - - - - 1,210

Alpha-fetoprotein (ng/mL) 21 6 5 5 4 6 4 5 4 5 3 3 3 3

PT-INR 1.09 1.17 1.13 1.13 1.15 1.23 1.24 1.0 1.0 1.0 1.1 1.0 1.0 1.0

White blood cells (mmc) 4,880 7,000 6,930 10,930 6,350 11,555 9,780 6,540 6,290 6,150 4,580 4,800 4,725 4,580

Hemoglobin (g/dL) 15.7 16.3 16.3 16.5 16.9 17.5 17.0 15.8 15.2 14.8 14.6 14.5 15.0 14.8

Platelet count (x109/L) 74 100 111 110 105 124 133 210 226 217 212 206 208 220

Liver Stiffness, kPa 17.6 14.5 10.1 11.9 16.5 7.7 10.9 - - - 10.1 - - -

CAP, dB/m - - - 222 297 - 268 - - - - - - -

Esophageal varices Small - Small - No - No - - - - - - -

Spleen length, cm 14.0 13.0 13.0 13.0 13.0 13.0 13.0 - - - 12.0 - - -

Creatinine (mg/dL) 0.82 0.83 0.88 0.85 1.00 0.90 0.86 0.80 0.74 0.73 0.72 0.74 0.76 0.74

Glycemia (mg/dL) /

Triglycerides (mg/dL) 118 / 129 119 / 91 119 / 122 111 / 71 120 / 69 122 / 56 111 / 59 128 / 142 89 / 172 115 / 193 104 / 89 109 / 126 99 / 101 96 / 106

Total Cholesterol / HDL

(mg/dL) 166 / 41 185 / 54 169 / 46 170 / 48 193 / 55 180 / 44 164 / 42 198 / - 198 / - 194 / - 172 / - 150 / - 189 / - 180 / -

4

5

BLV dose: In Patient 2, BLV dose was reduced from 10 to 5 mg/day at week 76; in Patient 3, BLV dose was reduced from 10 to 5 mg/day at 6

week 108 and to 2 mg/day at week 128. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline 7

phosphatase; GGT, gamma-glutamyl transferase; pCHE, pseudo-cholinesterase; CAP, Controlled Attenuation Parameter. Reference values: AST, 8

10–33 U/L; ALT, 6–41 U/L; ALP, 35–104 U/L; GGT, 5–36 U/L; Total bilirubin, 0.12–1.10 mg/dl; pCHE, 4,200–11,250 U/L; Albumin, 3.4–9

4.8 g/dl; IgG, 700-1600 mg/dL; INR, 0.84–1.20; White cells, 4,800–10,800/mmc; Hemoglobin, 12–16 g/dl; Platelet, 130–400 x109/L; 10

Journ

al Pre-

proof

20

Creatinine, 0.5–1.0 mg/dl; Glycemia, 70-110 mg/dL; Triglycerides, <150 mg/dL; Total cholesterol, <190 mg/dL; HDL, >45 mg/dL. 1

Journ

al Pre-

proof

All patients were on TDF treatment

HDV RNA ALT

HDV RNA ALT

HDV RNA ALT

3 patients with HDV compensated cirrhosis

+ contraindications to Interferon treatment

started entry-inhibitor

Bulevirtide (BLV) at 10 mg/day

as per compassionate use

HDV RNA undetectable in all

patients

Virological and biochemical response

maintained over 3 years of BLV even after

dose reduction

Regression of small esophageal varices

Recovery of HDV-related autoimmune

features

No adverse events

Supplementary Fig. 1: Graphical Abstract

Journ

al Pre-

proof

Figure

Patient 1 Patient 2

SF

U/1

05 c

ells

0

50

100

0

50

100

0

50

100

0

50

100

-8 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 1200

50

100

-8 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120

0

50

100

ZZ

ZM

0

1

2

3

4

5

6

7

30/1

1/20

17

31/1

2/20

17

31/0

1/20

18

28/0

2/20

18

31/0

3/20

18

30/0

4/20

18

31/0

5/20

18

30/0

6/20

18

31/0

7/20

18

31/0

8/20

18

30/0

9/20

18

31/1

0/20

18

30/1

1/20

18

31/1

2/20

18

31/0

1/20

19

28/0

2/20

19

31/0

3/20

19

30/0

4/20

19

31/0

5/20

19

30/0

6/20

19

31/0

7/20

19

31/0

8/20

19

30/0

9/20

19

31/1

0/20

19

30/1

1/20

19

31/1

2/20

19

31/0

1/20

20

29/0

2/20

20

31/0

3/20

20

30/0

4/20

20

31/0

5/20

20

30/0

6/20

20

31/0

7/20

20

31/0

8/20

20

30/0

9/20

20

31/1

0/20

20

30/1

1/20

20

HD

V R

NA

, Lo

g IU

/mL

ALT

, IU

/L

50

100

150

200

250

350

250

012 16 20 24 28 32 36 40 44 48 52 56 60 76 80 84 88 92 96 104 112 128120 144

HDV RNA

ALT

136-8 0 2 4 8 64 68 72

TDF 245 mg/24 h

35

3.9

4.5

<10

<160

112

4.1

4.4

TND

-

112

4.0

-

TND

-

106

4.0

4.2

<10

<160

141

4.0

4.2

TND

-

34

4.0

4.1

<10

-

171

3.9

-

<10

<160

68

3.9

-

<10

-

48

3.9

-

<10

-

88

3.9

3.8

<10

<160

96

3.9

-

TND

-

25

3.9

-

TND

-

193

3.9

3.8

<10

<160

229

3.8

-

TND

-

85

3.9

-

TND

-

97

3.9

3.7

TND

TND

168

3.9

-

TND

-

79

3.8

-

TND

-

177

3.8

-

TND

-

24

3.8

-

<10

-

55

3.8

3.7

TND

TND

43

3.7

-

TND

-

25

3.7

-

<10

-

43

3.7

3.7

<10

TND

168

3.8

3.6

<10

<10

63

3.7

-

<10

-

50

3.6

3.4

TND

TND

53

3.7

-

TND

-

70

3.7

-

<10

-

24

3.6

3.3

TND

TND

weeks

77

3.7

3.4

TND

TND

Normal ALT: <41 IU/L

BLV 10 mg/day BLV 5 mg/day

204,000392,000

239,000

104,000

4,050

642

115

35

17

<6TND

<6<6

211

232

211

115

40 3726 25

3232 29 3324 22 24 22 25 29 31

20

34 33 28 28 27 26 2632

20 23

TND TND TND TND TND TNDTND TND TND TND TND TND TND TND TND TND TND TND TND TND

24 22 20

350

Bile Acids µmol/L

HBsAg Log IU/mL

HBcrAg Log U/mL

HBV DNA IU/mL

HBV RNA cp/mL

HBV IFN-γ

T cells

HDV IFN-γ

T cells

T cells

against PMA

0

50

100

0

50

100

0

50

100

0

50

100

-8 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 1200

50

100

-8 0 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120

0

50

100

ZZ

ZM

68 60 52 44 36 28 20 12 4

0

1

2

3

4

5

6

7

30/1

1/20

17

31/1

2/20

17

31/0

1/20

18

28/0

2/20

18

31/0

3/20

18

30/0

4/20

18

31/0

5/20

18

30/0

6/20

18

31/0

7/20

18

31/0

8/20

18

30/0

9/20

18

31/1

0/20

18

30/1

1/20

18

31/1

2/20

18

31/0

1/20

19

28/0

2/20

19

31/0

3/20

19

30/0

4/20

19

31/0

5/20

19

30/0

6/20

19

31/0

7/20

19

31/0

8/20

19

30/0

9/20

19

31/1

0/20

19

30/1

1/20

19

31/1

2/20

19

31/0

1/20

20

29/0

2/20

20

31/0

3/20

20

30/0

4/20

20

31/0

5/20

20

30/0

6/20

20

31/0

7/20

20

31/0

8/20

20

30/0

9/20

20

31/1

0/20

20

30/1

1/20

20

HD

V R

NA

, Lo

g IU

/mL

ALT

, IU

/L

50

100

150

200

250

350

250

012 16 20 24 28 32 36 40 44 48 52 4 8 24 28 32 36 40 44 48 52 60 7668 92

HDV RNA

ALT

84-8 0 2 4 8 2 12 16 20

BLV 10 mg/day

TDF 245 mg/48 h

weeks

262318

Normal ALT: <41 IU/L

13,655

<6

2416

266

2,065

12,846

6,947

2,555

421 431421 455395

<6<6<6<6

1,312

601891

108

353

44,640

23,600

54,400

21,500

8,6206,360

1,410

378

75

13

26

20

156140

123122

94

57

39

24 20 25

125

20

47

24

54

74

108

333

97

70

89

58

37 3434 36 35 31

31

18 2130

7

10

2.8

TND

<160

42

12

<2

TND

-

40

11

<2

TND

-

43

13

<2

TND

<160

56

24

-

TND

-

165

15

-

TND

-

166

14

<2

TND

<160

71

21

-

TND

-

15

23

-

TND

-

25

21

<2

TND

<160

53

23

-

TND

-

46

20

-

TND

-

54

29

-

TND

<160

36

23

<2

TND

TND

10

15

<2

TND

TND

19

15

<2

TND

TND

13

13

-

TND

-

12

11

<2

TND

TND

14

1.5

<2

TND

TND

8

0.4

2.3

TND

TND

10

0.2

<2

TND

TND

10

0.2

-

TND

-

8

0.3

<2

TND

TND

8

0.4

-

TND

-

12

2

-

TND

-

-

0.6

<2

TND

TND

-

0.6

-

TND

-

-

0.5

<2

TND

TND

6

0.4

-

TND

-

6

0.5

<2

TND

TND

8

0.5

-

TND

-

Bile Acids µmol/L

HBsAg IU/mL

HBcrAg Log U/mL

HBV DNA IU/mL

HBV RNA cp/mL

8

0.5

-

TND

-

8

0.5

-

TND

-

87

26

96

A B

D E

HBV IFN-γ

T cells

HDV IFN-γ

T cells

T cells

against PMA

SF

U/1

05 c

ells

Journ

al Pre-

proof

0

1

2

3

4

5

6

7

30/1

1/20

17

31/1

2/20

17

31/0

1/20

18

28/0

2/20

18

31/0

3/20

18

30/0

4/20

18

31/0

5/20

18

30/0

6/20

18

31/0

7/20

18

31/0

8/20

18

30/0

9/20

18

31/1

0/20

18

30/1

1/20

18

31/1

2/20

18

31/0

1/20

19

28/0

2/20

19

31/0

3/20

19

30/0

4/20

19

31/0

5/20

19

30/0

6/20

19

31/0

7/20

19

31/0

8/20

19

30/0

9/20

19

31/1

0/20

19

30/1

1/20

19

31/1

2/20

19

31/0

1/20

20

29/0

2/20

20

31/0

3/20

20

30/0

4/20

20

31/0

5/20

20

30/0

6/20

20

31/0

7/20

20

31/0

8/20

20

30/0

9/20

20

31/1

0/20

20

30/1

1/20

20

HD

V R

NA

, Lo

g IU

/mL

ALT

, IU

/L

50

100

150

200

250

350

250

012 16 20 24 28 32 36 40 44 48 52 56 60 76 80 84 88 92 96 116 128124 144

HDV RNA

ALT

1360 4 8 64 68 72

TDF 245 mg/24 h

weeks

Normal ALT: <41 IU/L

BLV 10 mg/day BLV 5 mg

TND TND TND TND TND TND TND TND

350

<10

4.0

4.6

<20

-

4.0

-

-

233

4.0

4.4

-

184

4.1

4.6

<20

218

4.1

4.2

-

150

4.0

4.4

-

200

4.0

4.4

<20

211

4.0

4.3

-

190

4.0

-

-

164

4.0

4.3

<20

190

3.9

4.3

-

179

4.0

4.1

-

179

4.0

3.9

<20

-

-

4.1

-

-

-

4.2

-

202

4.0

4.0

TND

-

-

4.2

-

-

-

4.0

-

-

-

4.2

-

-

-

-

-

212

4.0

4.1

<20

-

-

-

-

195

3.9

-

-

202

3.9

4.2

<20

246

4.2

4.1

TND

251

4.0

3.9

-

132

3.9

-

-

162

3.9

-

<20

152

4.0

-

<20

168

3.9

-

-

132

3.9

-

TND

Bile Acids µmol/L

HBsAg Log IU/mL

HBcrAg Log U/mL

HBV DNA IU/mL

237

3.9

3.9

TND

BLV 2 mg

108

212

3.9

4.0

-

100

104,80364,575

33,394

18,321

1,812

704

12283

714

<6

7

3141

<6 <6

244

194

97

524651

34 35

35

34 28 33 28 33 30 30 30 30 32 32 29 31

<6 <6

192

37 39

34<6 <6 <6 <6 <6

TND TND TND TND TND

36 34 3731 30 33 29 23

22 22

154

3.8

-

-

174

3.9

-

-

139

3.9

-

-

Figure

Patient 3 C

Journ

al Pre-

proof