Droplet digital PCR assay provides intrahepatic HBV cccDNA quantification tool for clinical application

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.

guidelines of the Declaration of Helsinki and was approved by the appropriate institutional ethics review committees of each institute.

Analysis of virological markers.
HBV DNA was extracted from 5 μl of mouse serum using SMITEST EX-R+D KIT (MEDICAL AND BIOLOGICAL LABORATORIES). A real-time PCR assay (Taqman) was used for DNA quantification. HBV DNA titers of the chimeric mice were measured by real-time PCR, as described previously [3] [4]. Hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) were measured by chemiluminescent enzyme immunoassays, using commercial kits (FUJIREBIO Inc., Tokyo, Japan), as described previously [2] [5]. The detection limits of the HBsAg and HBcrAg assays are 0.005 IU/ml and 3 logU/ml, respectively. The HBV DNA titers of the patients were measured using a TaqMan polymerase chain reaction assay (COBAS TaqMan, ROCHE MOLECULAR SYSTEMS [lower detection limit: 20 IU/ml]) or Abbott RealTime HBV assay (lower detection limit: 10 IU/ml).

Isolation of genomic DNA from the livers of chimeric mice.
Genomic DNA was isolated from liver tissue using the phenol/chloroform method, as described previously [6]. Briefly, 50 mg of mouse liver were lysed with 5 ml of lysis buffer containing 10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 10 mM NaCl, 0.5% SDS and 500 ng of proteinase K at 37 o C overnight with gentle rotation. After incubation, an equal volume of Tris-saturated phenol was added and then rotated for 30 min at room temperature. The lysate was then clarified by centrifugation at 2,500 rpm for 10 min at 4 o C and the 4 supernatant was extracted twice with the phenol and once with chloroform: isoamyl alcohol. Approximately 0.1 volume of 3M sodium acetate and 2.5 volumes of pre-cooled ethanol were added to the DNA sample, followed by incubation at -80 o C for 1 hour and centrifugation at 3,000 rpm for 15 min at 4 o C. The precipitate was washed with 70% ethanol and resuspended in 300 µl TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
Protein free-DNA extraction from the mouse liver was carried by a modified Hirt extraction procedure [7]. Briefly, 100 mg of mouse liver were lysed in 4.5 ml of 10 mM Tris-HCl (pH 7.5), 10 mM EDTA and 0.6% SDS. After 30 min incubation at room temperature, the lysate was transferred into a 15 ml tube followed by addition of 1.2 ml of 5 M NaCl and incubation at 4 o C overnight. The lysate was then clarified by centrifugation at 10,000 rpm for 30 min and extracted twice with Tris-saturated phenol and once with phenol: chloroform. DNA was precipitated with two volumes of ethanol overnight at room temperature. After centrifugation at 4,000 rpm at 4 o C for 30 min, the DNA was washed with 75% ethanol and resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
The genomic DNA and protein free-DNA isolated from the livers of chimeric mice with HBV infection was treated with DNase-free RNase and digested with a restriction enzyme (Hind Ⅲ) before being analyzed.

Quantification of HBV cccDNA and relaxed circular (rc) DNA by ddPCR.
For HBV cccDNA quantitation of the liver DNA extracts, 1 µg of total liver DNA was treated for 30 minutes at 37°C with 10 U of plasmid-safe ATP dependent DNase (PSAD) (EPICENTRE, Madison, Wisconsin, USA) to digest single-stranded DNA and linear double-strand DNA. PSAD digestion was carried out in a 50 µl reaction containing 5 µl 10x PSAD buffer, 2 µl 25 mM ATP, 1 µl PSAD, 33 µl deionized water and 9 µl (20-1000 ng) total liver DNA. DNA samples pre-digested with PASD were used as template inputs for either qPCR or ddPCR amplification. Specific primers for cccDNA amplification (targeted across the single-stranded (SS) gap region of relaxed circular HBV DNA (rcDNA) were designed as shown in Fig. 1A  ddPCR and Southern blot (SB) analysis were compared using DNA isolated from two chimeric mice (mouse #1 without ETV and mouse #2 with ETV) in the chronic phase of HBV infection (Supplementary Fig. S4). As shown in Fig. S4A, the amounts of cccDNA detected by ddPCR were 1185.5 and 1428.5 copies/20 ng. SB assay loaded side-by-side on the same gel for direct comparison of both non-linearized and linearized Hirt extracted DNA (mouse #1 without ETV and mouse #2-4 with ETV). The non-linearized Hirt extracted cccDNA and protein-free rcDNA (Xho1-) were observed around 2.1 kb and 3.5 kb, respectively. The linearized Hirt extracted DNA with Xho1 (Xho1+), both bands around 2.1 kb and 3.5 kb migrated together at 3.2 kb. Importantly, the amounts of cccDNA detected by ddPCR were well-correlated with the cccDNA content (Xho1-) detected by the SB assay ( Supplementary Fig. S4A and B; mouse #1 without ETV and mouse #2 with ETV). The copy numbers of cccDNA per hepatocyte were examined by correcting the cccDNA content by the amount of a single copy gene, hRPP30 ( Supplementary Fig. S4C, D, and E).
Because only human hepatocytes harbor hRPP30 in these human hepatocyte chimeric mice, cccDNA/hepatocyte could be calculated by dividing the amount of cccDNA by hRPP30 divided by 2 ( Supplementary Fig. S4E). The intrahepatic cccDNA levels in the two HBV infected chimeric mice were 446.7 and 398.0 copies/20 ng, while the levels of hRPP30 in the corresponding mice were 346.7 and 335.3 copies/20 ng, indicating that, on average, one hepatocyte in these mice harbored approximately 2.37-2.57 copies of cccDNA. In contrast, cccDNA was barely detectable in non-infected mice (0.9 copies/20 ng) ( Supplementary Fig. S4C), although the level of hRPP30 was similar (499.3 copies/20 ng) to that in the HBV-infected mice.      (195-500) in VR with HBsAg clearance (B). Comparison between the groups was performed using the Mann-Whitney test.