Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients

Fanny Lebossé,Judith Fresquet,Fleur Chapus,Audrey Diederichs,Kamal Hamed,Clothilde Miaglia,Maëlle Locatelli,Fabien Zoulim,Aurore Inchauspé,Barbara Testoni

doi:10.1038/s41598-020-78001-1

Abstract

Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long-term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Long-term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all-but-one negative samples. Low preC/pgRNA level in telbivudine-treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No difference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof-of-concept clinical trials aiming at a functional cure of chronic hepatitis B.

Highlights

Hepatitis B virus (HBV) covalently closed circularDNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal
Baseline serum HBV DNA and ALT levels tended to be higher for HBe antigen (HBeAg)( +) patients and there was no significant difference between the two groups in the duration of telbivudine treatment (Table 1)
In the context of the global HBV cure research programs[15], the evaluation of the intrahepatic closed circular DNA (cccDNA) amount and its transcriptional activity will be instrumental for the assessment of the efficacy of the novel antiviral strategies under clinical investigation and for the development of non-invasive biomarkers reflecting the pool of cccDNA15

Summary

Introduction

Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Abbreviations ALT Alanine aminotransferase cccDNA Covalently closed circular DNA CHB Chronic hepatitis B ChIP Chromatin immunoprecipitation ddPCR Droplet digital PCR HBeAg Hepatitis B e Antigen HBsAg Hepatitis B s Antigen HBV Hepatitis B virus HCC Hepatocellular carcinoma HIV Human immunodeficiency virus NUCs Nucleos(t)ides analogues. PgRNA Pre-genomic RNA preC/pgRNA PreCore and pre-genomic RNA PTMs Post-transcriptional modifications qPCR Quantitative PCR (polymerase chain reaction) RCA Rolling circle amplification rcDNA Relaxed circular DNA ULN Upper limit of normal. Acetylation of cccDNA-associated histone tails has been correlated in vivo to patients’ viremia levels and intrahepatic cccDNA transcriptional activity[8,9], while the association of histone H3 trimethylation of lysine 9 and 27 with viral transcription levels in vivo is more controversial[9,10]

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Conclusion