Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection

Abstract

Background and aims: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints.

Approach and results: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA.

Conclusions: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

Figure 1.. Screen for antibodies cross-reactive with WHV precore/core antigens.

(A) Amino acid sequence alignment of the HBV genotype D (gt D) and WHV strain 7 (WHV7) precore/core proteins. The epitopes for each mAb are indicated by the dashed boxes. mAbs 1A11 and 7E9 are specific for the HBV and WHV eAg and PreC proteins. mAbs 19C18, 10E11, and T2221 react with the NTD shared by the HBV and WHV core and precore proteins. mAb A701 is selective for the non-phosphorylated (HBc and WHc) CTD whereas 14–2 for the phosphorylated (HBc and WHc) CTD. mAbs 366–2 (relatively non-selective for phosphorylation state) and 25–7 (selective for non-phosphorylation) are specific to HBc CTD without cross-reactivity to WHV proteins. The arrows indicate the putative cleavage sites in the production of WHeAg (WHe0 and WHe1), WHV PreC protein (WPreC0 and WPreC1), and HBeAg and HBV PreC (e1 and PreC1) as identified in this study. Two N-glycosylation motifs, NIT and NDT, in WHV (but not HBV) precore are underlined. (B) Immunoblot analysis of HBcrAg components in the serum of the genotype D HBV-infected chimpanzee (#1616, Wk22) and a WHV7-infected woodchuck by SDS-PAGE in a regular gel (12.5 cm in height). WHV-infected woodchuck serum samples from M1002 prior to wIFN-α treatment (Wk-3) and after recovery following treatment (Wk15) were included here as the WHeAg-positive and WHeAg-negative controls. (C) Schematic representations of each WHcrAg or HBcrAg species detected in the serum of WHV-infected woodchucks or HBV-infected chimpanzees shown in B. SP, signal peptide. g, glycosylated.

Figure 2.. WHeAg was N-glycosylated in WHV-infected woodchuck sera.

(A) Serum from a chronically infected woodchuck F6005 or an acutely infected woodchuck M7392 was treated with PNGase F followed by SDS-PAGE (12.5 cm gel) and immunoblotting using the anti-precore mAb 7E9 and the anti-NTD mAb T2221. (B) Immunoblot analysis of WHcrAg components in WHV-infected woodchuck sera following SDS-PAGE in a 32 cm height, high-resolution gel. Sera from acute WHV-infected woodchucks (M7392, F7386, and F7394; weeks refer to the time after WHV inoculation) and chronic WHV-infected woodchucks (F1018 and M7994; weeks refer to time after the beginning of antiviral treatments - these woodchucks had been inoculated as neonates and chronic WHV infection lasted for over a year before the start of the treatments), with or without PNGase F treatment, were analyzed. M7392 Pre (lane 1, 2, 15, 16), serum from the M7392 before infection, and M7994 Wk9 (lane 13, 14, 27, 28), serum from chronically infected M7994 after recovery following GS-9620 treatment, were included as negative controls. (C) Schematic representations of the various WHcrAg species detected by immunoblots shown in A & B. *, unknown woodchuck serum proteins cross-reactive with the anti-precore mAbs, 7E9 and 1A11, which co-migrated with the putative glycosylated WPreC proteins (g-WPreC0 and g-WPreC1).

Figure 3.. Different density profiles of WHeAg between chronically and acutely infected woodchucks.

Analysis of WHV virions and antigens by CsCl density gradient fractionation of the chronically and acutely WHV-infected woodchuck serum, F6005 and F7386, respectively. Gradient fractions were resolved by SDS-PAGE (regular height gel) and analyzed by immunoblotting using mAbs 7E9 and T2221. *, unknown woodchuck serum proteins cross-reactive with the anti-precore mAb 7E9, which co-migrated with the putative g-WPreC0 and/or g-WPreC1.

Figure 4.. Correlations between WHeAg and cccDNA, serum WHV DNA, and WHsAg.

Correlations of serum WHeAg with intrahepatic cccDNA (A), serum WHV DNA (B), and serum WHsAg (C). Serum samples and paired liver biopsies (n = 46) from 7 chronic WHV-infected woodchucks from the wIFN-α treatment study were included (15). Serum WHV DNA was measured by qPCR, WHsAg was measured by ELISA, and WHeAg was quantified by immunoblotting of the N-glycosylated WHeAg (g-WHe) and WHeAg (WHe) bands in the current study. The correlation coefficient was calculated by Spearman’s correlation test. Two-tailed p-value was calculated for a 95% confidence interval. The dashed grey line represents the detection limit of WHeAg (120 ng/ml) by our assay.

Figure 5.. Analysis of serum WHeAg and WHV DNA kinetics in woodchucks treated with different therapies.

(A) Changes of serum WHV DNA, WHeAg, WHsAg, and intrahepatic WHV cccDNA from chronically infected woodchucks treated with wIFN-α relative to week −3 (pre-treatment baseline). Chronically infected woodchucks were treated three times a week with wIFN-α at the indicated doses for 15 weeks total, as described (15). (B) Changes of serum WHV DNA, WHeAg, and WHsAg from chronically infected woodchucks treated with the TLR7 agonist GS-9620 relative to week −2 (pre-treatment baseline). Chronic WHV-infected woodchucks were treated with GS-9620 at the indicated doses three times a week for four weeks total (M7883 and F7979) or once a week for 8 weeks (M7969 and M7994) as described (17). Woodchuck sera samples resolved by SDS-PAGE and analyzed by immunoblotting using mAbs 7E9, or 1A11, and 19C18, are shown as the examples. (C) Changes of serum WHV DNA, WHeAg, and WHsAg from chronically infected woodchucks treated with telbivudine relative to week −1 or 0 (pre-treatment baseline). Chronic WHV-infected woodchucks were treated with telbivudine at a daily dose of 10 mg/kg for 12 weeks. WHc was detected by SDS-PAGE and immunoblotting using 19C18 but not 7E9 or 1A11 whereas WHeAg and PreC were detected by all those mAbs, as annotated in B. Dashed horizontal lines indicate the lower limits of detection. Dashed vertical boxes indicate the different phases of viral clearance: black, clearance of viremia alone; grey, clearance of viremia and antigenemia, as well as intrahepatic cccDNA.

Figure 6.. HBcrAg species in the sera of HBV-infected chimpanzees.

(A) Immunoblot analysis of HBcrAg components in the genotype D HBV-infected chimpanzee sera, resolved by SDS-PAGE in a high-resolution gel, using mAbs 1A11 (eAg/PreC specific), T2221 (NTD-specific), and 366–2 (CTD-specific) (Fig. 1A). (B) Analysis of serum HBV particles in infected chimpanzees by NAGE. Serum HBV particles from infected chimpanzees at the indicated time points (weeks or wk post-infection) were resolved by NAGE and transferred to nitrocellulose membrane. HBV DNA, capsid, envelope proteins (HBsAg particles co-migrating with virions) in the virions or subviral particles were detected sequentially using a ³²P-labeled HBV DNA probe, indicated HBc mAbs, and anti-HBs antibody, respectively, on the same membrane. V, virions containing either rcDNA or empty; S, viral surface or envelop proteins. Dashed boxes indicate the different phases of viral clearance involving distinct mechanisms during the resolution: black, viremia drop alone; grey, drop of viremia and antigenemia. (C) and (D) Correlations between intrahepatic cccDNA with serum HBc and HBcrAg. Serum samples and paired liver biopsies were from chimpanzees 1603 (Week 22), 1616 (Weeks 10, 22, and 38), 1618 (Weeks 12 and 18), A2A007 (Weeks 13 and 21), AOA006 (Week 5). Serum HBc and HBcrAg was quantified by immunoblotting. cccDNA quantification was normalized to mitochondrial DNA of each samples. The correlation coefficient was calculated by Spearman’s correlation test. Two-tailed p-value was calculated for a 95% confidence interval.

Figure 7.. HBcrAg species, biogenesis, and application as biomarkers to monitor HBV gene expression and replication during natural resolution.

(A) The HBV replication cycle, from virus entry to secretion, is shown schematically. Following virus entry into hepatocytes and cccDNA formation in the nucleus, pgRNA and subgenomic viral mRNAs are transcribed from the cccDNA. During translation of the 3.5 kb precore mRNA, the N-terminal signal peptide of the precursor protein p25 is cleaved by the ER-associated signal peptidase during translocation to the ER lumen, resulting in the production of p22 in the lumen, which then traffics through the secretory pathway, where the CTD of p22 is cleaved off by preprotein convertases such as furin, to generate HBeAg secreted into the blood. Other than HBeAg, the PreC protein, retaining the signal peptide but having the CTD cleaved, is also secreted into the blood. Due to rapid processing of the precore precursor p25 to the intracellular p22 or the secreted PreC proteins, p25 may not actually exist or be detectable in the cell. On the other hand, pgRNA is the template for translation of HBc and viral polymerase. pgRNA binds to viral polymerase and both are packaged into the assembling capsid formed by HBc, a process known to be blocked by IFNα. A delayed silencing effect on HBV gene expression by IFNα was also observed in some cases (dashed line). pgRNA is subsequently reverse transcribed into rcDNA and the resulting mature nucleocapsid is enveloped by the viral envelop proteins (L-HBs, M-HBs, and S-HBs) to form virions for secretion. The reverse transcription step is targeted by NUC treatment. Empty virions, which composes HBs and HBc, are secreted to the blood at ca. 100-fold excess over DNA virions. The current HBcrAg assay detects HBeAg, PreC protein, and HBc from complete and empty virions. (B) Summary of potential clearance mechanisms that likely contributed to peripheral viral marker clearance, as characterized in the current study. *, HBsAg may continue to be expressed from the integrated HBV DNA.