Optimization and validation of ddPCR for the detection of HBV cccDNA.
In the HBV genome, the incomplete plus strand has a variable 3’- end but a defined 5’- end at the position of 1592 near DR2, while the complete minus strand has defined 5’- and 3’- ends with a terminal redundancy of 9 bases. There is a gap at the position of 1826 near DR1 (Fig. 1A and B). To discriminate between the relaxed circular DNA (rcDNA) and the cccDNA of the HBV genome, we designed the primer to bind the negative strand on one side of the gap, and the probe to bind the same strand on the other side. Therefore, the forward and reverse primers, 5’-ACGGGGCGCACCTCTCTTTACGCGG-3’ [nt: 1519-1543] and 5’-CAAGGCACAGCTTGGAGGCTTGAAC-3’ [nt: 1862-1886], were designed against the minus and plus strand gaps of rcDNA and selected to specifically amplify DNA fragments from replication intermediate cccDNA but not from viral genome DNA. A TaqMan probe, which binds to DNA when PCR is performed, is composed of FAM and MGB quencher (Fig. 1B). To establish an assay to detect HBV cccDNA using ddPCR technology, we used total DNA extracted from the livers of human-liver-chimeric mice that had been infected with HBV (Fig. 1). Digestion of DNA by a restriction enzyme is believed to improve template accessibility . To test the effect of restriction enzyme digestion on the efficacy of cccDNA detection, temperature gradient ddPCR was performed in the presence of Hae III with fifteen restriction sites in the HBV genome outside the cccDNA amplification region on the sample DNA treated with PSAD. Xho I, which does not recognize the cccDNA amplification region but cuts at one site in the HBV genome, was used for comparison. As shown in Supplementary Fig. S1A and B, Hae III digestion provided better separation of positive and negative signals, and the results were less affected by the temperature as the signal intensity of positive droplet populations started to decrease at the annealing temperature below 58.5℃ for Xho 1 and 55.3℃ for Hae III. Based on these results, we decided to use Hae III digestion and set the annealing temperature at 61.2℃.
ddPCR accurately measures less than two-fold differences in cccDNA content.
To determine the performance of the ddPCR method for HBV cccDNA quantification, the plasmid containing the HBV genotype C2/Ce DNA genome (AB246345) was serially diluted from 105 to 101 copies/assay and determined by ddPCR. As a results, the cccDNA-specific ddPCR showed an excellent linear correlation between expected and observed HBV cccDNA copies/assay (R2=0.9997).
To compare the sensitivity and accuracy of cccDNA detection between ddPCR and qPCR, we serially diluted total DNA extracted from the livers of HBV-infected chimeric mice from 20 to 0.156 ng/assay and conducted ddPCR and qPCR. As shown in Fig. 2A, the results of both ddPCR and qPCR displayed high goodness of fit (R2=0.9935 and R2=0.9685, respectively). In addition, the results of ddPCR and qPCR exhibited a near-perfect correlation (R2=0.9958, p=2.12x10-70) (Fig. 2B). These results suggest that both ddPCR and qPCR are highly sensitive and sufficiently accurate to detect 2-fold differences of the intrahepatic cccDNA. In contrast, when the samples were diluted to various concentrations, ranging between 5 and 8 ng/assay, ddPCR but not qPCR was able to accurately measure the differences (R2=0.9416 and R2=0.8963, respectively) (Fig. 2C). In addition, ddPCR was found to more accurately detect less than 2-fold difference than qPCR in other assay ranges (R2= 0.9980 and R2=0.9972, respectively at higher assay range;12-20 ng/assay and R2=0.9974 and R2=0.9946, respectively at lower assay range; 4-6 ng/assay).
Additionally, the sensitivity of cccDNA detection was compared between ddPCR and qPCR. Linear HBV DNA fragments (nt 1372-2187) containing cccDNA-specific forward and reverse primer regions digested by NcoI and EcoRI from HBV genotype C2/Ce DNA genome (AB246345) were analyzed with and without PSAD treatment and conducted qPCR and ddPCR assays. When the linear HBV DNA in all dilution series (ranging 107-105 copies/assay) without PSAD treatment was analyzed by the cccDNA specific primer-probe set, both ddPCR and qPCR was able to accurately measure total DNA (cccDNA and HBV DNA containing rcDNA and ssDNA) (R2=0.998 and R2=0.998, respectively). In contrast, when the serially diluted samples were measured by the cccDNA specific primer-probe set with PSAD, ddPCR was able to accurately measure cccDNA at a concentration of approximately 1/45 of total DNA in all dilution series (R2=1), but qPCR was measure cccDNA at a concentration of approximately 1/32 of total DNA in all dilution series (R2=1). These results suggest that the fine sensitivity of cccDNA-specific ddPCR, excluding linear HBV-DNA containing the amplicon and integrated cccDNA to 1/45 after PSAD treatment. The limits of quantitative value (LOQ) and detection value (LOD) for HBV-cccDNA-specific ddPCR were determined using serial dilutions of total DNA extracted from the livers of HBV-infected and non-infected chimeric mice (Fig. 2E). The LOD and LOQ in mouse liver were 1 copy/ ddPCR assay and 20 copies/ ddPCR assay, respectively, and those were 5.9 copies/qPCR assay and 60.2 copies/qPCR assay, respectively. Collectively, these results suggest that both ddPCR and qPCR detect intrahepatic cccDNA with high sensitivity, but ddPCR is more accurate than qPCR in measuring less than two-fold differences.
Specific detection of cccDNA by ddPCR.
The specificity of cccDNA detection was compared between ddPCR and qPCR. Serially diluted total DNA extracted from the sera of HBV-infected chimeric mice, from 0.32 to 0.04 µl/assay, was analyzed by ddPCR and qPCR. Several studies reported that total DNA in the sera and the culture medium could contain a large amount of HBV DNA and a small amount of cccDNA [19, 20, 21], indicating that the cccDNA-specific ddPCR of the serum should generate a very low signal. As references, the serum HBV DNA levels of the HBV-infected chimeric mice were measured using a primer-probe set that could detect both cccDNA and rcDNA (herein referred to “set 1”) . As shown in Fig. 2D, the results of both ddPCR and qPCR using the set 1 primer-probe set showed high concordance (R2=0.9994 and R2=0.9906, respectively). When these serum samples were analyzed by the cccDNA specific primer-probe set without PSAD treatment, both ddPCR and qPCR was able to accurately measure cccDNA at a concentration of approximately more than 1/600 of total DNA in all dilution series, ranging between 0.32 and 0.04 µl/assay (R2=0.9022 and R2=0.9601, respectively). Moreover, when the serial diluted serum samples were measured by the cccDNA specific primer-probe set with PSAD treatment, ddPCR was able to accurately measure cccDNA at a concentration of approximately 1/1000 of total DNA in all dilution series, ranging between 0.32 and 0.04 µl/assay (R2=0.9915), but qPCR was unstable at low cccDNA concentrations (R2=0.9259, Fig. 2D). Because expressing uPA in the liver of the uPA/SCID mice with human hepatocytes induces a sufficiently damaged liver (Tateno et al., PLoS One, 2015), we suggest that the cccDNA in hepatocytes could flow out to the blood and the cccDNA could be detectable in sera of the chimeric mice. Unfortunately, although we performed Southern blotting (SB) experiments, the mice serum volumes are too small to detect any signals after PSAD treatment; we were unable to provide data whether the detected signal was cccDNA or rcDNA in false-positive by ddPCR.
Therefore, we tried to determine the cccDNA in sera from 7 inactive HBV carries with normal liver function using the HBV cccDNA-specific ddPCR with PSAD treatment. The assay was conducted in triplicate. The cccDNA signal was undetectable in all samples with HBV-DNA levels up-to 5.1 log IU/mL, suggesting that the specificity of the ddPCR assay for cccDNA was 100% (0/21), and the assay could detect cccDNA in mice serum. Finally, we compared ddPCR and SB analysis using DNA isolated from two chimeric mice in the chronic phase of HBV infection with or without ETV treatment (Supplementary Fig. S2). As shown in the Supplementary Information, these values were closely correlated with the cccDNA content detected by the SB assay.
Collectively, these results suggest that ddPCR could detect intrahepatic cccDNA with higher specificity and sensitivity than qPCR, depending on the cccDNA-specific ddPCR, especially after PSAD treatment.
The intrahepatic HBV cccDNA level correlates strongly with serum HBV parameters.
To determine the extent to which the intrahepatic cccDNA level is reflected by the HBV markers in the serum, the cccDNA contents were measured in 24 HBV-infected chimeric mice during 2 conditions of infection; spreading phase/partially infected mice (n=8, low HBV group) and stable phase/fully infected mice (n=16, high HBV group), and then correlated with the serum HBsAg and HBcrAg levels in each condition. As shown in Fig. 3A and B, the intrahepatic cccDNA levels were more significantly correlated with both the serum HBsAg level (R2=0.5639, P=3.5442x10-5) and the HBcrAg level (R2=0.6703, P=2.0217x10-4) in HBV high group compared with the low HBV group (R2=0.4091, p=0.00745 and R2=0.7093, p=0.01912, respectively), due to the small number of mice in Low HBV levels group. These results suggest that both HBcrAg and HBsAg are excellent surrogate serum markers of intrahepatic cccDNA.
Hepatocyte proliferation has little impact on the total amount of cccDNA in the liver.
Having established a highly quantitative cccDNA-specific ddPCR assay, we examined the impact of hepatocyte proliferation on the stability of cccDNA. All six chimeric mice undergoing hepatocyte repopulation were inoculated with HBV (1x106 copies per mouse) 4 weeks after transplantation, at which time the reconstitution rate was less than 30%, based on serum human albumin (hALB) levels (Fig. 4A) . As shown in table S1, the 6 mice derived from this experiment were indicated with an asterisk (week4#1: *, week4#2: †, week10 ETV #1: ‡, week10 ETV #2: §, week10 control #1: ||, week10 control #2: ¶, respectively.). Serum HBV DNA levels were measured in all mice 4 weeks later, ranging between 1.7 to 6.8 x105 copies/ml (Fig. 4B). Two chimeric mice were sacrificed on week 4 and two of the remaining four were administered ETV orally at 0.2 mg/kg/day for 4 weeks. The other two mice served as controls. The HBV DNA levels had already decreased after one week of ETV treatment and continued to decrease during the treatment, reaching a nadir on week 10 (1.6 log10 copies reduction) (Fig. 4B). In the ETV-treated chimeric mice, serum HBsAg and HBcrAg levels peaked at week 6 and week 5, respectively, and then decreased slightly (Fig. 4C and D). These markers continued to increase in the control group during the same period (Fig. 4B, C and D). Serum human albumin levels increased in both groups (Fig. 4E), reflecting liver repopulation, but the body weights did not change (Fig. 4F). As shown in Fig. 4G and H, HBV rcDNA, ssDNA and cccDNA were readily detectable in the control mice at week 10, but not in the ETV treated mice, indicating the low sensitivity of SB.
We then analyzed the levels of intrahepatic cccDNA at weeks 4 and 10 after HBV infection (i.e. immediately before the ETV treatment (week 4) and two weeks after the termination of ETV treatment (week 10)) (Fig. 4I). Before ETV treatment, 96.0-142.0 copies of cccDNA were detected in 1,000 ng of total liver DNA. Interestingly, almost the same amounts of cccDNA were detected in the ETV-treated mice at week 10 after HBV infection, consistent with the serum HBcrAg and HBsAg levels. In contrast, cccDNA levels in the control mice increased markedly to 9,800 and 19,800 copies/1,000 ng), a greater than 100-fold increase in 6 weeks. To determine the amount of cccDNA/hepatocyte, intrahepatic hRPP30 was quantified at the corresponding time points. As shown in Fig. 4J, the hRPP30 level increased by approximately 2.6 and 2.3-fold in the ETV-treated and control mice, in 6 weeks. The results are consistent with the increase of serum hALB levels (Fig. 4E) and suggest the ongoing human hepatocyte repopulation of the chimeric mouse livers. Consequently, the cccDNA level per hepatocyte decreased from 0.016 and 0.024 copies/cell before ETV treatment to 0.011-0.012 copies/cell after ETV treatment (Fig. 4K), while it increased to 1.361 and 1.886 copies/cell in the control mice. Collectively, these results suggest that the total amount of cccDNA in the liver is not affected by hepatocyte repopulation, although the cccDNA level/hepatocyte appears to be reduced after ETV treatment.
Intrahepatic HBV cccDNA in patients with HBV-associated hepatocellular carcinoma.
To apply this ddPCR-based method to clinical samples, we measured the amount of intrahepatic cccDNA and RPP30 in tumor and non-tumor tissues from five HBV-related HCC patients using the ddPCR assay. As shown in Table 1, varying levels of intrahepatic cccDNA were detected in three HBsAg-positive patients among the five HBV-related HCC patients. In two HBsAg-positive, HBcrAg-positive patients (patients 3 and 4), the cccDNA levels were 0.00933-0.05081 copies/cell in non-tumor tissues, and 0.93442-1.17975 copies/cell in tumor tissues. In the HBsAg-positive, HBcrAg-negative patient (patient 2), cccDNA was detected weakly at 0.00013 copies/cell in tumor tissue, while it was undetectable in non-tumor tissue. In two HBV-related HCC patients whose HBV infections had resolved, cccDNA levels were below the detection limit. Intrahepatic RPP30 in all 6 HCC patients, including the negative control, were 36,100-109,500 and 51,650-104,000 copies/1000 ng in non-tumor and tumor tissues, respectively. These data showed that ddPCR could detect cccDNA in tumor and non-tumor tissues.
The impact of PEG-IFN treatment of patients with HBeAg-negative CHB on intrahepatic cccDNA levels.
To investigate the effect of PEG-IFN on intrahepatic cccDNA, we determined the levels of intrahepatic cccDNA in patients with HBeAg-negative CHB at baseline and 48 weeks after PEG-IFN treatment (Fig. 5, Supplementary Fig. S3 and Table S2). Thirteen patients with HBeAg-negative CHB who received 48 weeks of PEG-IFN monotherapy were categorized into 3 groups: non-VR, VR without HBsAg clearance and VR with HBsAg clearance. At baseline, there was no significant difference in the levels of HBV DNA, HBsAg, and HBcrAg in the serum, or the levels of total cccDNA and cccDNA per cell in the liver, among the three groups (Supplementary Table S2). As shown in Fig. S3 and Table S2, serum HBV DNA levels in the non-VR group either did not change or decreased temporarily at the end of PEG-IFN treatment, only to rebound thereafter. In the VR without or with HBsAg clearance groups, HBV DNA levels decreased continuously after PEG-IFN treatment (Supplementary Fig. S3). Serum HBsAg levels in the non-VR and VR without HBsAg clearance groups were 2.8±1.1 (1.32-3.26) and 1.1±0.9 (0.40-2.38) log IU/mL, respectively, at week 48, whereas HBsAg levels were undetectable at weeks 12 and 24 in the VR with HBsAg clearance group (Supplementary Fig. S3). Serum HBcrAg levels in the non-VR group could be quantitated (4.3±1.4 (2.8-6.6) Log U/mL), while those in the VR without and with HBsAg clearance groups were mostly under the quantitation limit at week 48 (Supplementary Fig. S3). Interestingly, the levels of intrahepatic cccDNA and cccDNA per cell were significantly lower in the VR groups than the non-VR group at week 48 (p=0.0160 and p=0.0432, respectively), associated with a significant reduction of cccDNA per cell (p=0.0051) (all data shown in Fig. 5A-F and Supplementary Table S2). These significant changes in cccDNA levels in the VR groups were associated with a higher peak of ALT than the non-VR group (p=0.0165) (Fig. 5A-G). In particular, in the VR with HBsAg clearance group, intrahepatic cccDNA levels at week 48 after PEG-IFN treatment were lower than those in the VR without HBsAg clearance group (p=0.036), in association with the higher ALT peak (p=0.060) (Fig. S4A-B), although HBcrAg levels in most VR patients were undetectable, due to the limited sensitivity. Remarkably, cccDNA levels could be readily quantitated even in patients (11 and 12) with HBsAg clearance after PEG-IFN treatment (Fig. 5C and Supplementary Table S2).