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Service d’hépatologie, Hôpital de la Croix Rousse, HospiceCls de Lyon, Lyon, FranceCentre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052, CNRS UMR 5286, Université Claude Bernard Lyon 1, Lyon, France
Service d’hépatologie, Hôpital de la Croix Rousse, HospiceCls de Lyon, Lyon, FranceCentre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052, CNRS UMR 5286, Université Claude Bernard Lyon 1, Lyon, France
Centre Hépato-Biliaire, Université Paris-Saclay, Unité Inserm 1193, Hôpital Paul-Brousse, Assistance Publique-Hôpitaux de Paris (AP-HP), Villejuif, France
Centre Hépato-Biliaire, Université Paris-Saclay, Unité Inserm 1193, Hôpital Paul-Brousse, Assistance Publique-Hôpitaux de Paris (AP-HP), Villejuif, France
Service de chirurgie générale et transplantation hépatique, Hôpital de la Croix Rousse, Hospices Civils de Lyon, Lyon, FranceCentre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052, CNRS UMR 5286, Université Claude Bernard Lyon 1, Lyon, France
Service d’hépato-gastro-entérologie, Hôpital Grenoble-Alpes, Grenoble, France ; (New Address : Service d’hépatologie, Hôpital Henri Mondor, Assistance Publique-Hôpitaux de Paris, Créteil, France)
Service de virologie, Hôpital de la Croix Rousse, Hospices Civils de Lyon, Lyon, FranceCentre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052, CNRS UMR 5286, Université Claude Bernard Lyon 1, Lyon, France
Service d’hépatologie, Hôpital de la Croix Rousse, HospiceCls de Lyon, Lyon, FranceCentre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052, CNRS UMR 5286, Université Claude Bernard Lyon 1, Lyon, France
Service d’hépatologie, Hôpital de la Croix Rousse, HospiceCls de Lyon, Lyon, FranceCentre de Recherche en Cancérologie de Lyon (CRCL), INSERM U1052, CNRS UMR 5286, Université Claude Bernard Lyon 1, Lyon, France
This is the first prospective real-life study investigating intrahepatic viral marker in transplanted patients
•
Viral genome was detected very early in the graft despite NUC and HBIG prophylaxis
•
At M12 post-OLT, HBV markers were detected in the liver graft or blood circulation in all but one patient with matched samples.
Abstract
Background and Aims
Prophylaxis with nucleos(t)ide analogues (NUC) and hepatitis B immunoglobulin (HBIG) has decreased the rate of Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT), but the duration of this prophylaxis remains debated. Our aim was to investigate the recurrence of both intrahepatic and serum HBV markers after OLT in patients receiving long-term NUC and HBIG prophylaxis.
Methods
31 HBV-positive patients benefiting from OLT were prospectively enrolled in 5 French centers between 2012 and 2015. Tissue samples from native liver, liver reperfusion biopsy, and 12 months post-OLT (M12) biopsy were collected. Intrahepatic HBV markers were quantified by droplet digital Polymerase Chain Reaction. Serum HBcrAg and HBsAg were quantified using the Lumipulse platform.
Results
Among the 31 patients, 26 were HBeAg-negative and 28 had undetectable serum HBV DNA at OLT. All patients received HBIG and NUC after OLT and serum HBV DNA was undetectable at M12. Twenty-six of the twenty-seven available native livers had detectable total (t)HBV DNA (median 0.045 copies/cell), 21/27 were positive for cccDNA (0.001 copies/cell), and 19/27 for HBV 3.5Kb-RNA (0.0004 copies/cell). Among the 14 sequential reperfusion and M12 biopsies, 7 were positive for HBV markers on the reperfusion sampling and 6 of them were also positive at M12. Of the 27 patients with available serum samples at M12, 8 were positive for HBcrAg and 5 for HBsAg by ultrasensitive quantification, although negative by conventional techniques. Overall, among the 17 patients having a matched biopsy and serum sample at M12, only one had undetectable HBV markers both in liver and serum.
Conclusions
Our results demonstrate a very early detection of viral genome in the graft and intrahepatic viral recurrence despite NUC and HBIG prophylaxis.
FV, FL, SR, BR, MJY, VL, ST, ML, SSA, CS and BT declare no conflicts of interest. FZ received consulting fees from: Aligos, Antios, Assembly, Gilead, GSK; and research funding to INSERM from: Assembly, Beam, Janssen. DS received consulting fees from Biotest, Go Liver, Gilead Sciences, Seabelife. RA received consulting fees from Gilead and research funding to CHU of Nice, France, from: Novartis. GPP received fees from Gilead. AH, MH are employees and stakeholders of Roche Molecular Diagnostics.
Financial Support statement
Gilead: unrestricted research grant to support hospital research staff involved in clinical data acquisition, clinical data monitoring, data base management, and biological sample collection and storage. Gilead had no implication in the study design, generation and interpretation of data, nor in the writing of the manuscript. Fujirebio: provided the Lumipulse HBsAg and HBcrAg quantification assays. French Agence Nationale de la Recherche (ANR): This study was supported by a public grant attributed by the French Agence Nationale de la Recherche (ANR) as part of the second “Investissements d’Avenir” program (reference: ANR-17-RHUS-0003) and by the European Union (grant EU H2020-847939-IP-cure-B) to FZ and ML. Agence Nationale de Recherches sur le SIDA, les Hépatites Virales et les Maladies Infectieuses Emergentes (ANRS MIE) grant ECTZ8323 to FZ, ML, and BT.
Authors’ contributions
Concept and design: FZ, BT, FV, SSA; methodology and investigation: FV, BT, FB, CS, AH; formal analysis and visualization: FV, BT; data curation: ST; writing – original draft: FV, BT, FZ; writing – review and editing: BT, FZ, ML, FL, DS, VL, MH, SR, BR, JYM, VL, ML, FZ, DS, RA and GPP; supervision and funding acquisition: FZ; resources: MH, FL, SR, BR, JYM, VL, ML, FZ, DS, RA and GPP.
All authors had access to the study data and reviewed and approved the final version of the manuscript
Data Transparency Statement
Deidentified individual participant data that underlie the reported results will be made available upon request 3 months after publication for a period of 5 years after the publication date.
Lay Summary
In this work, we show that despite the recommended prophylaxis based on NUC and HBIG, HBV can infect the new liver very rapidly after transplantation. Twelve months after transplantation, the majority of patients had at least one HBV marker detected either in serum or the liver. Therefore, our results demonstrate early intrahepatic viral recurrence despite NUC and HBIG therapy and underline the importance of an optimal patient compliance to the antiviral prophylaxis to prevent viral rebound.
Introduction
Chronic hepatitis B (CHB) affects 296 million people worldwide and is the main cause of hepatocellular carcinoma (HCC)
. Current CHB treatments, based on nucleos(t)ide analogues (NUC), are effective in decreasing viral load, but are curative in less than 5% of cases. Orthotopic liver transplantation (OLT) represents the definitive treatment for decompensated CHB cirrhosis, complicated or not by HCC. Currently, CHB is a common indication for OLT and represents 30% of all OLT performed in Asia and 10% in Europe and in USA
Historically, CHB was considered a contraindication due to the high risk of HBV recurrence after OLT, resulting in graft losses and poor survival rate of less than 40% at 5 years
. The prophylaxis regimen used to prevent HBV recurrence has changed over the last 3 decades with the advent of Hepatitis B immunoglobulins (HBIG) and different generations of NUC
. Currently, by combining 3rd generation NUC and HBIG perfusions, the rate of HBV recurrence after OLT, defined by HBsAg positivity in the serum, is less than 10%
Long-term management of the successful adult liver transplant: 2012 practice guideline by the American Association for the Study of Liver Diseases and the American Society of Transplantation.
2020 position statement and recommendations of the European Liver and Intestine Transplantation Association (ELITA): management of hepatitis B virus-related infection before and after liver transplantation.
. Because of the high cost of the administration of HBIG by intravenous (IV) infusion or subcutaneous (SC) injection, reducing the HBIG prophylaxis duration is considered in specific patient populations based on the exclusion of risk factors such as pre-OLT viral load, Hepatitis D Virus (HDV) co-infections and when optimal patient compliance to treatment is ensured
2020 position statement and recommendations of the European Liver and Intestine Transplantation Association (ELITA): management of hepatitis B virus-related infection before and after liver transplantation.
Several studies have reported the use of NUC monotherapy without HBIG for the prophylaxis of HBV recurrence. These studies reported a rate of detectable HBsAg in serum of approximately 8-10% after a follow-up of 2 to 8 years post-OLT, which was associated with maintained viral suppression and the absence of virologic relapse when using NUCs with high barrier to resistance, and overall a very good survival rate
. In these patients, the detection of HBsAg indicates that NUC monotherapy does not fully prevent reinfection of the graft. Indeed, it has been shown in hepatocyte culture models that NUC inhibit the viral replication but do not prevent neither covalently closed circular (ccc) DNA formation in de novo infected hepatocytes nor HBV genome integration and HBsAg production
Inhibitory Effect of Adefovir on Viral DNA Synthesis and Covalently Closed Circular DNA Formation in Duck Hepatitis B Virus-Infected Hepatocytes In Vivo and In Vitro.
Hepatitis B Virus DNA Integration Occurs Early in the Viral Life Cycle in an In Vitro Infection Model via Sodium Taurocholate Cotransporting Polypeptide-Dependent Uptake of Enveloped Virus Particles.
None of the previously mentioned studies on simplified prophylaxis have investigated HBV intrahepatic markers, and only a few retrospective studies have analyzed HBV recurrence on liver biopsies
Serum hepatitis B virus DNA before liver transplantation correlates with HBV reinfection rate even under successful low-dose hepatitis B immunoglobulin prophylaxis.
. In a retrospective study of 12 patients with a 60-month follow-up, under double prophylaxis, cccDNA was quantifiable in 9 of them (including 3/3 who had a quantifiable serum viral load at OLT)
. Another investigation including 25 patients transplanted for HBV receiving dual NUC-HBIG prophylaxis has reported the detection of intrahepatic total (t)HBV DNA and cccDNA 3 years after OLT in 87% and 17% of samples, respectively, and a recurrence of serum HBsAg in 2 (8%) patients
. In another study including patients receiving lamivudine prophylaxis after OLT, 2/44 (4.5%) patients were positive for tHBV DNA but none for cccDNA after an average of 88 months; 56% of these patients had received HBIG prophylaxis
. In another retrospective study, 12/20 patients were positive for both cccDNA and serum HBcrAg after OLT, and the levels of both markers were positively correlated
Significance of hepatitis B virus core-related antigen and covalently closed circular DNA levels as markers of hepatitis B virus re-infection after liver transplantation: HBV markers after liver transplantation.
. Overall, these studies were heterogeneous regarding the timing of viral markers analysis after OLT and did not assess the recurrence of HBV infection at early time points. Moreover, more sensitive quantification technologies such as droplet digital Polymerase Chain Reaction (ddPCR) assays have been developed since then, which could allow a more precise appraisal of HBV recurrence on the graft
A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.
Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
In this context, the primary objective of the ECOGREFFE prospective cohort study was to analyze HBV DNA (cccDNA and tHBV DNA) and RNA presence in the native liver and the graft with more sensitive assays to gain insight into the kinetics of HBV recurrence, in patients receiving the recommended long-term NUC and HBIG prophylaxis in France. The secondary objective was to investigate the correlation between intrahepatic viral molecular markers and novel serum HBV biomarkers.
Patients and methods
Prospective cohort
Patients over 18 years, with or without HCC, and listed for OLT were eligible for inclusion in the French prospective multicenter study “ECOGREFFE” (Clinical Trial number: NCT02602847) between 2012 and 2015 in 5 French OLT centers: Lyon-Croix Rousse, Villejuif-Paul Brousse, Montpellier, Nice, and Grenoble. The present study is an ancillary study to the “ECOGREFFE” study. The inclusion criteria were: being included in the “ECOGREFFE” study, having benefited from an OLT for an indication of HBV-related disease. Medical information and laboratory results were collected prospectively. Three patients with positive HDV serology were excluded from the analysis.
Whenever possible, a liver sample from native HBV-infected liver (27 patients), from reperfusion biopsy (26 patients) and at 12 months after OLT (M12) (17 patients) were collected. In parallel, blood samples were collected at the time of registration on the OLT waiting list (30 patients), at M3 (29 patients), and at M12 (27 patients). Additionally, according to ECOGREFFE study protocol, 6 samples from patients transplanted for an alcoholic-related disease or HCV-related disease at the Lyon-Croix Rousse liver transplant center were used as technical negative controls. Liver samples were snap frozen and all serum and tissue samples were stored at -80° until processing. For each patient, the clinical follow-up duration was 12 months post-OLT. The study was conducted in accordance with the relevant ethical standards and approved by the local advisory committee (IRB: CPP Sud-Est IV [ref: A-12-158]; ID RCB: 2012-A00383-40).
Clinical virological tests
Conventional laboratory tests
Serum viral load was quantified in each center using either the Roche (Roche Diagnostics, Mannheim, Germany) or the Abbott assay (Abbott Diagnostic, Chicago, United States), with detection limit of 10 IU/mL and 20 IU/mL, respectively. The conventional HBsAg quantification was carried out by the conventional methods in each center using either Roche (Roche Diagnostics) or Architect Abbott assay (Abbott Diagnostic), with a limit of quantification set at 50 mIU/mL.
Ultra-sensitive HBsAg quantification
Quantitative levels of HBsAg at OLT registration time, at M3, and at M12 were determined using the Lumipulse G HBsAg assay (Fujirebio Europe, Gent, Belgium) on the Lumipulse G600II Analyzer (Fujirebio) according to the manufacturer’s instructions. HBsAg levels were expressed in mIU/mL and the assay measurement linear range spanned from 5 mIU/mL to 150 000 mIU/mL. Samples for which the HBsAg level was <5 mIU/mL were considered negative. Samples for which the HBsAg level was ≥150 000 mIU/mL were diluted with a manufacturer-supplied dilution reagent and retested to quantify HBsAg values.
HBcrAg quantification
The serum hepatitis B core-related antigen (HBcrAg) quantification assay simultaneously measures the concentration of denatured HBeAg, HBcAg, and the precore protein p22cr (aa - 28 to aa 150). The quantitative levels of HBcrAg from patients’ serums were determined using the Lumipulse G HBcrAg assay (Fujirebio) on the Lumipulse G600II Analyzer (Fujirebio) according to the manufacturer’s instructions. HBcrAg levels were expressed in logU/mL. The lowest sensitivity limit of the machine was 2 logU/mL, but a specificity of 100% is reached for values ≥ 2.8 logU/mL
. Therefore, samples for which the HBcrAg level was ≥ 2 and <2.8 logU/mL were considered negative. Samples for which the HBcrAg level was ≥ 2.8 and < 3 logU/mL were considered detectable but not quantifiable. Samples for which the HBcrAg level was ≥7 logU/mL were diluted with a manufacturer-supplied dilution reagent and retested in order to quantify HBcrAg values.
Circulating HBV RNA quantification
Circulating HBV RNA (cirB-RNA) was quantified by real-time PCR using the Roche HBV RNA investigational assay (Roche Diagnostics, Pleasanton, USA) for use on the Cobas 6800/8800 Systems (Roche Diagnostics)
. The assay is a nucleic acid quantitative test performed with EDTA plasma or serum sample, for which the lower level of quantification (LLoQ) is 10 copies/mL (linearity range 10 to 109 copies/mL on armored RNA template) and the lower level of detection (LLoD) is 3 copies/mL
Allweiss L, Testoni B, Yu M, Lucifora J, Ko C, Qu, B, et al. Quantification of the Hepatitis B Virus cccDNA minichromosome: lessons learned from a concerted harmonization initiative. Submitted 2022.
Quantification of total HBV DNA, cccDNA, and 3.5-kb-RNA in liver samples by ddPCR
The quantification of the absolute copy numbers of intrahepatic tHBV DNA, cccDNA, and 3.5-kb-RNA was performed using the QX100TM Droplet DigitalTM PCR System (Bio-Rad, Hercules, USA) according to the manufacturer’s instructions and detailed in Supplementary data file.
Statistical analysis
All data were analyzed using using Prism Graphpad version 8.1.2 (GraphPad Software, San Diego, USA). Data were expressed as median [interquartile range (IQR)] for continuous variables and as count (percentage) for categorical variables. Statistical correlations were tested using the Pearson test for normal variables and the Spearman test for other variables. Categorical variables were compared using the Chi-square or Fischer’s exact tests and quantitative variables were compared using the Student t-test or non-parametric tests (Mann-Whitney or Kruskall-Wallis tests), when appropriate. The significance α threshold was set at 0.05.
Results
Patient characteristics
A total of 54 patients were initially screened at the time of registration on the OLT waiting list between 2012 and 2015: 31 of them underwent an OLT for CHB and were included in the present prospective study. The median [IQR] age at the time of OLT of HBV patients was 57.3 [49.4-63.4] years, and 27 (87%) were males. At OLT registration, 22 (71%) patients had an HCC, five of them had an active HCC at the time of OLT. The median [IQR] delay between HBV diagnosis and OLT was 8.8 [3.0-15.5] years. Before OLT, 30/31 (97%) patients had an antiviral treatment (for a median [IQR] duration of 28.7 [13.2-75.9] months), with most of them receiving Entecavir (38%) or Tenofovir (38%) (Table 1). Out of the 31 patients, 3 had detectable serum viral load at the time of OLT (median [IQR] value at 175 [131-334] IU/ml) (Table 1). No patient was co-infected with Human Immunodeficiency Virus (HIV) or Hepatitis C Virus (HCV) and three patients with HDV positive serology were excluded from the study. Additionally, 6 HBV-negative transplanted patients were included as technical negative controls: 3 were transplanted for alcohol-related disease and 3 for chronic hepatitis C (all were negative for HBV markers in liver and serum; data not shown). All graft donors were anti-HBc negative.
Table 1Patients’ characteristics at the time of orthotopic liver transplantation
Clinical characteristics
HBV group (n=31)
Age at OLT, median [IQR] (years)
57.3 [49.4-63.4]
Male/female sex ratio, n (%)
27 (87.1%)/ 4 (12.9%)
BMI, median [IQR] (kg/m2)
24.0 [22.1-26.8]
MELD score at OLT registration, median [IQR]
11.1 [6.1-21.3]
CHILD-PUGH score at OLT registration, median [IQR]
B7 [A5-B9]
A, n (%)
13 (41.9%)
B, n (%)
10 (32.3%)
C, n (%)
8 (25.8%)
Waiting time on the waiting list, median [IQR] (days)
North Africa includes Tunisia, Libya, Morocco, Egypt and Algeria. Africa refers to other countries on the continent, not included in the previous list.
North Africa includes Tunisia, Libya, Morocco, Egypt and Algeria. Africa refers to other countries on the continent, not included in the previous list.
2 (6.5%)
Others
2 (6.5%)
HCC (presence)
22 (71%)
Number of nodules, median [IQR]
2.2 [1.0-2.8]
Size of the largest nodule, median [IQR] (mm)
24.2 [15.0-30.5]
Serum AFP level, median [IQR] (μg/L)
5.0 [3.0-12.8]
Patient with active HCC at LT time
5/22
Patient with HCC treated before LT
20/22
•
History of chemoembolization
12
•
History of surgical resection
6
•
History of Thermo-ablation
5
•
History of other treatment
3
Virological status at the time of OLT
8.8 [3.0-15.5]
2 (6.5%)
3 (9.7%)/ 28 (90.3%)
175 [131-334]
Delay between HBV diagnosis and OLT, median [IQR] (months)
HBeAg positive, n (%)
Serum viral load (detectable/undetectable), n (%)
Level of detectable serum viral load, median (IU/mL)
HBV treatment before OLT, n (%)
Entecavir
13 (38.2%)
Tenofovir
13 (38.2%)
Lamivudine
3 (8.8%)
Tenofovir + Emtricitabine
1 (2.9%)
None
1 (2.9%)
Duration of antiviral treatment before OLT, median [IQR] (months)
28.7 [13.2-75.9]
AFP: Alpha-fetoprotein; BMI: Body Mass Index; HBV: Hepatitis B Virus; HCC: hepatocellular carcinoma; HDV: Hepatitis D Virus; IQR: Interquartile Range; MELD: Model for End-Stage Liver Disease; OLT: Orthotopic Liver Transplantation.
1 North Africa includes Tunisia, Libya, Morocco, Egypt and Algeria. Africa refers to other countries on the continent, not included in the previous list.
The M12 survival was 100%. All patients received a dual prophylaxis regimen of NUC and HBIG: all patients had IV HBIG administration during the anhepatic phase and during the first week after OLT. After the first week, 22/31 (71%) patients were administered HBIG intravenously and 9/31 (29%) subcutaneously (Table 2). Median [IQR] levels of anti-HBs antibodies at 3 and 12 months after OLT were 825.0 mIU/ml [629.5 - >1000.0] and 487.0 mIU/ml [395.3 - >1000], respectively. As immunosuppressive regimen, 61% of the patients received Tacrolimus and Mycophenolate Mofetil, around 13% of patients received Tacrolimus and Everolimus or Tacrolimus alone and only one patient received Cyclosporin alone. Three patients received other drug regimens (Table 2). All patients were negative for serum HBsAg with conventional assays at M3 and M12. Serum HBV DNA was undetectable at M3 and M12 in all patients (Table 2). A total of 4 patients underwent a second OLT: two for a primary graft non-function, one for ischemic cholangiopathy, and one for a “small for size” syndrome. No re-transplantation was performed for HBV recurrence. Median [IQR] delay before the first and second LT was 26.5 days [16.0 – 83.5]. Two biopsies at M12 were from these retransplanted patients.
At M12, liver function tests were in the normal range for the majority of patients and only 3 had increased Gamma-glutamyl Transferase (GGT) in the context of obesity (Table 2).
Quantification of HBV markers in liver samples
At the time of OLT, 27 biopsy samples of liver explant were available from the 31 HBV-positive patients. Among these samples, 26/27 (96%) were positive for tHBV DNA, 21/27 (77%) for cccDNA, and 19/27 (74%) for 3.5-kb HBV RNA. The median [IQR] tHBV DNA, cccDNA, and 3.5-kb HBV RNA levels were 0.077 [0.009-0.19] copies/cell, 0.002 [0.0009-0.007] copies/cell, and 0.012 [0.003-0.05] copies/cell, respectively. The median [IQR] cccDNA transcriptional activity, estimated as the 3.5-kb-HBV RNA/cccDNA ratio in the explanted livers, was 8.38 [0.5-36.8] (Fig. 1).
Figure 1Evolution of tHBV DNA (A), cccDNA (B), 3.5-kb-HBV RNA (C), and cccDNA transcriptional activity (D) on liver explant, reperfusion biopsy and at 12 months after orthotopic liver transplantation. Red dots: patients with detectable serum HBV DNA before OLT. Mann-Whitney test was used to compare the groups, α threshold=0.05. LLoQ: Lower Level of Quantification; OLT: orthotopic liver transplantation.
The reperfusion biopsies were collected within a median [IQR] delay between portal vein anastomosis and biopsy of 48 minutes [41 – 62]. Seven out of 26 reperfusion biopsies available (27%) were positive for tHBV DNA, and the median [IQR] concentration was 0.005 [0.00007-0.03] copies/cells (Fig. 1A). At M12, among the 17 liver biopsy samples available, 9/17 (53%) were positive for tHBV DNA, and the median [IQR] concentration was 0.00045 [0.0002-0.0008] copies/cells (Fig. 1A).
Regarding cccDNA quantification, 11/26 (42%) reperfusion biopsies were positive, and the median [IQR] concentration was 0.001 [0.0001-0.011] copies/cells (Fig. 1B). At M12, 8/17 (47%) biopsy samples were cccDNA-positive, and the median [IQR] concentration was 0.0004 [0.0001-0.003] copies/cells (Fig. 1B).
3.5-kb HBV RNA were detected in 2/26 (8%) reperfusion biopsies, and the median [IQR] concentration was 0.0011 [0.001-0.016] copies/cells. At M12, 6/17 (35%) biopsy samples were 3.5-kb RNA-positive, and the median [IQR] concentration was 0.00006 [0.00003-0.006] copies/cells (Fig. 1C). Given the very low intrahepatic viral load after transplantation, only 1 sample was positive for both cccDNA and 3.5-kb HBV RNA on the reperfusion biopsy and 3 samples at M12, and thus could not be used for the calculation of cccDNA transcriptional activity (Fig. 1D).
Quantification of intrahepatic HBV markers in longitudinal samples
In 14 patients, native liver sample, sequential reperfusion and M12 biopsies were available. All of them had quantifiable HBV markers, either DNA, RNA or both, in the native liver. Seven patients out of 14 (50%) had quantifiable HBV markers on the reperfusion sampling and 6 of them (86%) were also positive at M12. Of the 7 patients with HBV-negative reperfusion biopsy, 4 (57%) presented quantifiable HBV markers at M12 post-OLT. Thus, only 3/14 patients remained negative for HBV markers quantification both at reperfusion and M12 post-OLT sampling. Overall, 10/14 (71%) patients had HBV-positive M12 post-OLT liver biopsy and six of them had already quantifiable levels of HBV markers in their reperfusion liver sample.
Ultra-sensitive HBsAg, HBcrAg quantification and circulating HBV RNA in serum
At the time of inclusion in the study (referred to as baseline), all 30 available samples were positive for serum HBsAg (determined using the Lumipulse assay), and the median [IQR] HBsAg concentration was 572,282 [67,374-1,156,666] mIU/mL. Additionally, 22/30 (73%) samples were positive for HBcrAg, and the median [IQR] concentration 4.2 [3.7-4.9] logU/mL. Serum HBV RNA level could be quantified in 12/30 (40%) samples and the median [IQR] concentration was 143.5 [27.7-498.8] copies/mL (Fig. 2).
Figure 2Evolution of serum HBsAg (A), HBcrAg (B), and HBV RNA (C) quantification before OLT, and at 3 and 12 months after orthotopic liver transplantation. Red dots: patients with detectable serum HBV DNA before OLT. Mann-Whitney test was used to compare the groups, α threshold=0.05. LLoQ: Lower Level of Quantification; OLT: orthotopic liver transplantation.
Serum HBV markers were then evaluated in samples collected at M3 and M12. At M3, 1/29 (3%) samples were positive for HBsAg (determined using the Lumipulse assay; 14.6 mIU/mL; Fig. 2A). At M12, while all samples were negative using conventional HBsAg assays, 5/27 (18%) patients had a quantifiable HBsAg determined using ultra-sensitive quantification with a median [IQR] concentration of 13.9 [11.0-15.1] mIU/mL (Fig. 2A).
At M3, 7/29 (24%) samples were positive for HBcrAg, with six of them having quantifiable values (median [IQR] concentration: 3.2 [3.1-3.3] logU/mL) (Fig. 2B). At M12, 8/27 (30%) samples were positive for HBcrAg; the median [IQR] concentration was 3.2 [3.1-3.2] logU/mL and was determined for the 5 patients with quantifiable values (Fig. 2B).
HBV RNA was undetectable in all serum samples analyzed at M3 and M12 (Fig. 2C).
Correlation between intrahepatic and serum viral markers at baseline
Pre-OLT baseline serum HBcrAg level was significantly correlated with the tHBV DNA (r=0.74; p<0.0001), cccDNA (r=0.61; p=0.0004), and 3.5-kb-HBV RNA (r=0.84; p<0.0001) levels in the native liver. Pre-OLT ultrasensitive serum HBsAg concentration was correlated with tHBV DNA (r=0.62; p=0.0008) and 3.5-kb-HBV RNA (r=0.55; p=0.0032) levels, but not with the intrahepatic cccDNA level (r=0.20, p=0.21) in native liver. Before OLT, circulating HBV RNA level was not correlated with cccDNA level (r=0.3090; p=0.055) and tHBV DNA (r=0.118; p=0.263) in native liver. cirB-RNA was correlated with intra-hepatic 3.5-kB HBV RNA (r=0.714; p=0.002) and with transcriptional activity of cccDNA (r=0.640; p=0.029) in native liver.
Association between intrahepatic and serum viral markers 12 months post-OLT
As stated above, 5/27 patients had a positive and quantifiable serum HBsAg levels using the ultrasensitive assay at M12, while being undetectable by conventional methods (Fig. 3A). M12 biopsies were available for 3 of these 5 patients; HBV DNA could be detected in all of them (3/3 for cccDNA, 2/3 for tHBV DNA) and 1 was also positive for 3.5-kb HBV RNA (Fig. 3A). The corresponding reperfusion biopsy samples were all negative for intrahepatic viral markers, except one which had quantifiable HBV DNA (both tHBV DNA and cccDNA) (Fig. 3A).
Figure 3Representation of ultra-sensitive HBsAg (A) or HBcrAg (B) positive patients according to HBV intrahepatic markers on reperfusion biopsy and at 12 months post-OLT. Each circle represents a liver sample at reperfusion or 12 months post-OLT. White circles: negative quantification for both HBV DNA and RNA; yellow circles: positive quantification for HBV DNA and negative for HBV RNA; red circles: positive quantification for both HBV DNA and RNA. Circles with shaded profile: biopsy not available. To be noted: HBV DNA indicates either tHBV DNA or cccDNA quantification. OLT: orthotopic liver transplantation.
HBcrAg was detectable in 8/27 samples at M12, with quantifiable values (>3 logU/ml) in 5 of them. Six out of these eight patients had a M12 biopsy available and 30% of them were positive for cccDNA detection (Fig. 3B). Six reperfusion biopsy samples out of these eight patients were available: all were negative except one, which had quantifiable levels of both cccDNA and tHBV DNA.
At M12, one patient (1/32) had quantifiable values for both ultrasensitive HBsAg and HBcrAg. The analysis of the reperfusion biopsy showed undetectable levels of HBV markers, while only cccDNA was detectable in the M12 biopsy.
We did not find any difference between the duration of antiviral therapy before OLT and HBV recurrence infection on the graft at M12, defined by tHBV DNA (p=0.26), cccDNA (p=0.38), 3.5-kb HBV RNA (p=0.25), HBsAg (p=0.24), or HBcrAg (p=0.25) levels. There was no correlation between HBV recurrence on the graft at M12 and anti-HBs antibody levels at M3 and M12, nor with the HBIG route injection type (SC or IV), the presence of active HCC at OLT or the type of immunosuppressive regimen. The two patients who underwent a second liver transplant had undetectable qHBsAg and HBcrAg in the serum, while one M12 biopsy had quantifiable intrahepatic HBV DNA and RNA.
Finally, among the 17 patients having a matched biopsy and serum sample at M12, only one had undetectable HBV markers in both the liver and serum compartments. These 17 patients did not differ from the 14 others (with no matched biopsy and serum sample at M12) on clinical characteristics at the time of OLT and on virological parameters on explant.
Discussion
The present study, based on the analysis of both cross-sectional and sequential serum and liver samples, showed that despite the undetectability of the conventional HBV markers in the serum, HBV recurrence can occur very early after OLT in a majority of patients receiving an anti-HBc negative graft.
During long-term NUC administration, intrahepatic and serum HBV DNA levels are decreased to very low levels, resulting in their undetectability with classical qPCR techniques in most cases
Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
. Nevertheless, qPCR-negative patients have been shown to experience viral relapse after treatment withdrawal, thus suggesting an undetected residual cccDNA pool in their liver
. At the time of OLT, in the present study, patients were receiving NUC for a median duration of 28.7 months and serum HBV DNA was undetectable for most of them. However, using a ddPCR assay, we were able to detect HBV nucleic acids in almost all available liver explants. These data are consistent with those of immunocompetent CHB patients under long-term NUC therapy viral suppression or those of patients with occult HBV infection
Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
. The intrahepatic cccDNA levels in the liver explants were slightly lower than those reported in non-transplanted CHB patients under long-term NUC therapy
Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
Thanks to the access to sequential liver samples after OLT in a subset of patients, we could investigate the time course of HBV recurrence on the graft using ddPCR assays. Interestingly, our results showed a very early recurrence of HBV in the grafted liver in half of reperfusion biopsy samples, despite IV HBIG perfusion during the operative period. Several hypotheses can be put forward to explain this early HBV recurrence. First, residual viral particles present in the blood because of incomplete viral suppression, or release from the liver during hepatectomy, or viral particles present in extrahepatic reservoirs during the anhepatic phase, may infect the new liver
. Indeed, it has been shown using experimental models that residual low viremia levels in a context of NUC therapy can be infectious in liver-humanized mice
Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.
Inhibitory Effect of Adefovir on Viral DNA Synthesis and Covalently Closed Circular DNA Formation in Duck Hepatitis B Virus-Infected Hepatocytes In Vivo and In Vitro.
Hepatitis B Virus DNA Integration Occurs Early in the Viral Life Cycle in an In Vitro Infection Model via Sodium Taurocholate Cotransporting Polypeptide-Dependent Uptake of Enveloped Virus Particles.
. Assuming that HBV could circulate just below the lower limit of detection of PCR assays in the NUC-suppressed patients awaiting OLT, up to 105 HBV copies could be present in the blood circulation at the time of surgery and may be thus available to infect the estimated 1011 hepatocytes of the graft. This remaining viral burden may not be fully neutralized by the infusion of HBIG during the anhepatic phase. It has also been shown that HBV mutants may escape both HBIG and NUC and infect the liver graft despite prophylaxis
; however, it is unlikely that this phenomenon contributed to the graft infection since serum HBV DNA remained non quantifiable by qPCR in all patients. Lastly, recent in vitro data indicated that formation of cccDNA in the nucleus of infected hepatocytes occurs rapidly after viral inoculation
, thus accounting for the possibility of a very early infection of the graft that could be already detectable at the time of the reperfusion biopsy.
At M12, cccDNA could be detected at very low levels in half of the available samples, and the associated cccDNA transcription rates were very low. Overall, at least tHBV DNA or cccDNA could be detected at M12 in the majority of samples, suggesting a significant rate of infection of the graft, which remained under control thanks to the antiviral prophylaxis protocol. Altogether, the results are consistent with the effectiveness of the HBIG and NUC prophylaxis in maintaining a strong antiviral pressure and suggest that very few hepatocytes might harbor cccDNA. Such a low viral burden in the liver graft would certainly not be detected by conventional immunostaining assays. The development of new technologies for single-cell genomic and proteomic analysis may allow further investigations to address this question
When analyzing novel serum viral biomarkers, we found that, at M12, 5 patients were positive for HBsAg quantified by an ultrasensitive method and 8 were positive for HBcrAg, although all patients tested negative for both serum HBsAg and HBV DNA with conventional assays. In these patients, the HBsAg concentrations remained very low and the anti-HBs antibody concentration was high at M3 and M12. A possible explanation for this observation could be the presence of immune complexes masking HBsAg or the presence of HBs mutants escaping HBs antibody recognition in the conventional assay
. Since patients received NUC therapy, serum HBV DNA remained undetectable by qPCR. The long-term consequences of HBsAg seropositivity after OLT are currently unknown. Ultrasensitive serum HBsAg quantification correlated with tHBV DNA and 3.5-Kb HBV RNA in the explanted liver, but not with cccDNA. This was consistent with the findings in immunocompetent CHB patients
Serum Hepatitis B core-related antigen (HBcrAg) correlates with covalently-closed circular DNA transcriptional activity in chronic hepatitis B patients.
. The significant number of patients with positive detection of serum HBcrAg at M12 was in contrast with the very low detection of intrahepatic 3.5-Kb RNAs, the only viral transcript species able to generate both HBe and HBc antigens. We cannot rule out that this could be due to limitations in sensitivity of intrahepatic RNA detection or in the specificity of the HBcrAg assay.
After OLT, in the few patients with a positive HBcrAg or HBsAg quantification, no obvious correlation was found with the levels of intrahepatic viral markers. This is in contrast to the findings by Matsuzaki et al. who found a positive correlation between serum HBcrAg and intrahepatic cccDNA levels determined by a less sensitive qPCR analysis after OLT; however, detailed information on the prophylaxis regimen was not reported
Significance of hepatitis B virus core-related antigen and covalently closed circular DNA levels as markers of hepatitis B virus re-infection after liver transplantation: HBV markers after liver transplantation.
One limitation of our prospective cohort study is that it was not designed as a comparative clinical trial of long-term versus short term HBIG. The early HBV infection of the liver graft tissue and the fact that HBsAg could be detected by an ultrasensitive method in the serum of some patients at M12, underline the importance of an optimal patient compliance to the antiviral prophylaxis to prevent viral rebound, regardless of the duration of HBIG administration.
In conclusion, this study investigating viral markers in both the liver and serum compartments on sequential samples showed that the recurrence of HBV infection can occur very early after the transplantation despite standard HBIG and NUC prophylaxis. Thus, our data strongly emphasize the importance of optimal patient compliance to antiviral therapy and raise the question of the dosage and duration of HBIG prophylactic regimen. Randomized studies would be warranted to specifically address the impact of HBIG prophylactic therapy versus HBIG-free regimens on early tissue reinfection and its correlation with clinical outcome.
Acknowledgments:
We thank Carrie NEWSOM for reviewing the manuscript, and all the medical, surgical and clinical research staff of the investigating centers who actively participated in the study.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
Long-term management of the successful adult liver transplant: 2012 practice guideline by the American Association for the Study of Liver Diseases and the American Society of Transplantation.
2020 position statement and recommendations of the European Liver and Intestine Transplantation Association (ELITA): management of hepatitis B virus-related infection before and after liver transplantation.
Inhibitory Effect of Adefovir on Viral DNA Synthesis and Covalently Closed Circular DNA Formation in Duck Hepatitis B Virus-Infected Hepatocytes In Vivo and In Vitro.
Hepatitis B Virus DNA Integration Occurs Early in the Viral Life Cycle in an In Vitro Infection Model via Sodium Taurocholate Cotransporting Polypeptide-Dependent Uptake of Enveloped Virus Particles.
Serum hepatitis B virus DNA before liver transplantation correlates with HBV reinfection rate even under successful low-dose hepatitis B immunoglobulin prophylaxis.
Significance of hepatitis B virus core-related antigen and covalently closed circular DNA levels as markers of hepatitis B virus re-infection after liver transplantation: HBV markers after liver transplantation.
A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.
Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.
Allweiss L, Testoni B, Yu M, Lucifora J, Ko C, Qu, B, et al. Quantification of the Hepatitis B Virus cccDNA minichromosome: lessons learned from a concerted harmonization initiative. Submitted 2022.
Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.
Serum Hepatitis B core-related antigen (HBcrAg) correlates with covalently-closed circular DNA transcriptional activity in chronic hepatitis B patients.
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