Molecular characterization of intrahepatic and extrahepatic hepatitis B virus (HBV) reservoirs in patients on suppressive antiviral therapy

Abstract

The hepatitis B virus (HBV) replicates via an error-prone reverse transcriptase generating potential drug-resistant quasispecies. The degree of HBV variability in liver vs peripheral blood mononuclear cells (PBMC) in patients on long-term suppressive antivirals is unclear. We characterized HBV replication, drug resistance and molecular diversity in patients with plasma HBV DNA undetectable by clinical assays. Explant liver (n=9), PBMC (n=6) and plasma (n=7) from nine such patients undergoing liver transplantation were evaluated for HBV genomes by sensitive PCR/nucleic acid hybridization assay. Cases with HBV DNA in liver and PBMC were tested for covalently closed circular DNA (HBV cccDNA). HBV polymerase (P) amplicons were cloned, sequenced and both P and overlapping surface (S) gene sequences were analysed. HBV DNA was detected in 43% (3/7) of plasma, 100% (9/9) of liver and 83% (5/6) of PBMC samples. HBV cccDNA was detected in all liver and one PBMC sample. Four patients had a clinical diagnosis of resistance. HBV P gene sequencing revealed 100% wild type (wt) in plasma (2/2), 83% wt in PBMC (5/6) but livers of 3/9 (33%) contained wt and 6/9 (66%) carried resistance to lamivudine and/or adefovir. The translated S gene revealed no changes affecting HBV antigenicity. Sequences from livers with antiviral resistant mutants revealed greater interpatient quasispecies diversity. Despite apparent HBV suppression, the liver continues to support HBV replication and extrahepatic HBV can be detected. PBMC may be a sanctuary for wt virus during antiviral therapy, while the liver harbours more drug-resistant viruses. Drug resistance correlates with intrahepatic viral diversity.

Fig. 1

HBV genome detection in liver and peripheral blood mononuclear cells (PBMC) collected from patients on suppressive anti-HBV therapy. (a) Detection of HBV P gene in explant liver (L) from nine patients (Cases 1–9). (b) Detection of HBV covalently closed circular DNA cccDNA in explant liver (L) and PBMC (P) from six patients (Cases 4–9). HBV DNA and cccDNA were detected using direct and nested PCR/nucleic acid hybridization (except in Cases 3 and 7 in which HBV DNA could be detected by a single round PCR). Serial dilutions of recombinant HBV DNA, used as a positive control, (10²–10⁶ virus genome equivalents, vge/mL) were simultaneously tested to determine approximate HBV viral load in liver samples by densitometry analysis. Negative controls included direct PCR water (DW), nested PCR water (NW), and a mock (M) extracted sample. The size of the expected direct (1140 bp) or nested (766 bp) DNA and cccDNA amplicons (144 bp) is indicated to the left of the figure.

Fig. 2

Detection of HBV P gene in plasma and PBMC of six patients (Cases 4–9) and HBV C gene in plasma of Case 5 by PCR/nucleic acid hybridization. All patients were on long-term suppressive antiviral therapy, and HBV DNA was undetectable in serum by clinical PCR assays. All reactions were run in parallel with strict contamination controls as described in the legend to Fig. 1. The positive control was recombinant HBV DNA. The molecular sizes of the expected HBV C (403 bp) and P (766 bp) gene fragments are indicated to the left of each panel.

Fig. 3

Detection of HBV P gene and HBV covalently closed circular DNA (cccDNA) amplicons in different compartments of HBV occurrence in a patient (Case 6) treated with emtricitabine (FTC) and tenofovir (TDF). The detection of HBV DNA in plasma (Plas), liver (Liv) and peripheral blood mononuclear cells (PBMC) by PCR/nucleic acid hybridization is illustrated, although HBV DNA was undetectable in serum by standard clinical assays. All reactions were run in parallel with strict contamination controls, as described in legend to Fig. 1. The size of the expected direct HBV P gene amplicons (766 bp) and the HBV cccDNA amplicons (144 bp) is indicated to the left of each panel.

Fig. 4

Sequence and phylogenetic analysis of HBV P gene in liver shows solely wild-type virus in Cases 1, 5, 7 and antiviral resistant (ART) mutations in Cases 2, 3, 4, 6, 8 and 9. There was a significant trend (P < 0.05) towards increased HBV quasispecies diversity in patients with antiviral resistant HBV quasispecies in liver compared to those with wild-type sequences. For each patient 10–20 HBV P gene clones were analysed.