Divergent preS Sequences in Virion-Associated Hepatitis B Virus Genomes and Subviral HBV Surface Antigen Particles From HBV e Antigen-Negative Patients

Divergent preS Sequences in Virion-Associated Hepatitis B Virus Genomes and Subviral HBV Surface... Abstract Background Hepatitis B virus (HBV) surface proteins (HBsAg) coat the viral particle and form subviral particles (SVPs). Loss of HBsAg represents a functional cure and is an important treatment goal. Methods We analyzed the impact of the HBV genotypes A–E and pre-S mutations on SVP expression in hepatitis B virus e antigen (HBeAg)-negative chronic HBV-infected patients. A HBV genome harboring a preS1-deletion was analyzed in hepatoma cells. Results We observed a genotype-specific ratio of the 3 surface proteins (SHBs/MHBs/LHBs), reflecting differences in the morphology and composition of SVPs. Deletions/mutations in the preS1/preS2 domain, detected in released viral genomes, did not affect the molecular weight of MHBs and LHBs in these patients. In contrast, LHB molecular weight was altered in vitro using an HBV genome harboring a preS1-deletion derived from one of these patients. Conclusion Differences in composition of SVPs may result in genotype-specific immunogenicity and pathogenesis. In the patients with preS-mutations, secreted HBsAg and released viral genomes cannot be derived from the same genetic source. As viral genomes are derived from covalently closed circular DNA (cccDNA), HBsAg is presumably derived from integrated DNA. This important HBsAg source should be considered for novel antiviral strategies in HBeAg-negative chronic HBV-infected patients. HBV, cccDNA, integrated DNA, HBsAg, filaments, spheres Despite the availability of an effective vaccine, chronic infection with the hepatitis B virus (HBV) is still a major cause of severe liver disease [1]. Its circular 3.2-kb genome contains 4 open reading frames, which encode the preC/Core protein, the regulatory X protein, the viral polymerase, and surface proteins [2]. The 3 surface proteins are encoded by 1 open reading frame containing 3 in-frame AUG start codons. Depending on the initiating site, large surface protein (LHBs; preS1, preS2, and s domain), middle surface protein (MHBs; preS2 and s), or small surface protein (SHBs; only s) are expressed [3]. The surface protein (HBsAg) coats the infectious viral particle (Dane particle). In addition, subviral particles (SVPs) are highly overproduced from HBV infected cells. These particles consist only of HBsAg and lipids and are divided according to their shape into filaments of variable length and 22-nm spheres. While spheres are released via the general secretory pathway, filaments like infectious viral particles are released via so-called multivesicular bodies [4, 5]. However, the role of SVPs in HBV-related pathogenesis is not fully understood. Although they are noninfectious, they have been reported to distract neutralizing antibodies from the virus, thereby allowing viral spread and persistence in the host [6]. Either nucleot(s)ide analogues (NA) or pegylated interferon, depending on viral load, presence of liver inflammation, and/or liver fibrosis, are recommended for antiviral treatment for HBV-related hepatitis [7–10]. While sufficient suppression of viral replication can be achieved with NA treatment, HBsAg loss as a functional cure is a rare event due to the presence of covalently closed circular DNA (cccDNA) and integrated DNA. The cccDNA is organized into a chromatin-like structure and can persist for decades in the human liver [11]. Integration of HBV DNA can occur through the entire host’s genome. However, so far no distinct integration sites have been identified in nontumor tissues [12]. Transcription of HBsAg from integrated DNA is still possible as the HBsAg open reading frame remains intact in most cases and retains its 2 native promoters [12]. Several new antiviral concepts targeting different sites of the viral life cycle, including HBsAg release, are currently in development [13]. Recently, it was reported that HBsAg serum levels are associated with HBV genotypes and frequent mutations in precore and preS in patients with HBV e antigen (HBeAg)-negative HBV infection [14, 15]. In the present study we extend our recent in vitro analysis [16] of HBV genotypes by studying the impact of HBV genotypes and mutations in the preS domain on the composition and morphology of SVPs in patients from a large European cohort of HBeAg-negative HBV-infected patients. MATERIALS AND METHODS Patient Samples Serum samples from HBeAg-negative patients (HBV DNA <100000 IU/mL, alanine transaminase <2 × upper limit of normal) infected with human HBV genotype A–E (GTA–GTE) from the German multicenter Albatros trial (listed at Clinical.Trial.gov: NCT01090531) were enrolled for analysis. The Albatros study is an observational long-term follow-up study of patients with HBeAg-negative HBV infection with low-level viremia who do not require antiviral therapy. Main inclusion and exclusion criteria are listed in the Supplementary material. Serum from these patients was collected and stored at −80°C. Quantitative serum HBsAg was measured with the Abbott Architect platform (Abbott Diagnostics) during clinical routine. The study was approved by local ethics committees and written informed consent was obtained from all patients. The study was performed in accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines. Sucrose Density Gradient Centrifugation Viral and subviral particles from patient sera were either enriched or separated via ultracentrifugation. For enrichment, 100–200 µL patient sera were layered on top of a cushion of 10% (w/v) d(+) sucrose dissolved in TN150 buffer and centrifuged in a SW60 rotor (Beckman Coulter, Krefeld, Germany) at 42000 rpm for 2.5 hours at 4°C; 40 µL of TN150 buffer was used for resuspension. For separation, 100–200 µL patient sera (GTA n = 9; GTB n = 7; GTC n = 4; GTD n = 12; GTE n = 2) were layered onto a linear gradient and centrifuged in an SW60 rotor at 32000 rpm for 18 hours at 4°C. The gradient was composed from bottom to top as follows: 40%, 37.5%, 35%, 32,5%, 30%, 27.5%, 25% (w/v) d(+) sucrose dissolved in TN150 buffer. SDS-PAGE and Western Blot Analysis Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were performed according to standard procedures [17]. The primary antibodies anti-β-actin (mouse, monoclonal, Sigma-Aldrich, Seelze, Germany), anti-transferrin (rabbit, monoclonal, Santa Cruz Biotechnology, Heidelberg, Germany), anti-SHBs (HB01, mouse, monoclonal, kindly provided by Aurelia Zvirbliene, Lithuania) [18], anti-LHBs (MA18/7, mouse, monoclonal, kindly provided by Dieter Glebe, Gießen) [3], and anti-preS2 (Q19/10, mouse, monoclonal, kindly provided by Dieter Glebe, Gießen) [19] were used. Peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). At least 10 samples of each genotype were enriched and equal protein amounts, determined via Bradford assay, were analyzed via western blot analysis. Plasmids HBV genomes of 1.5-fold length, derived from 2 HBeAg-negative HBV GTA-infected patients were used: (1) preS1 deletion of 45 nucleotides at positions 2944–2988; and (2) no mutation or deletion regarding the preS1/preS2 domain [14]. Transient Transfection Huh7.5 cells were transiently transfected using 1 µg DNA and 6 µL linear polyethyleneimine (Polysciences Europe GmbH, Hirschberg, Germany) and grown for 48 or 72 hours. HBsAg ELISA HBsAg quantities were analyzed in fractions of sucrose density gradient centrifugation using the Enzygnost HBsAg 6.0 ELISA (Siemens HealthCare Diagnostics, Erlangen, Germany) according to the manufacturer’s instructions and were specified as signal to cutoff ratios. Real-Time PCR Quantitative real time polymerase chain reaction (qPCR) was performed using a custom-designed TaqMan gene expression assay (Thermo Fisher Scientific, Darmstadt, Germany), described in the Supplementary material. Primers Primers used in this study are listed in Supplementary Table S1. Electron Microscopy Samples were incubated with carbon-coated nickel grids for 3 minutes followed by fixation with formaldehyde. Washed grids were negatively stained with a 2% aqueous uranyl acetate solution for 10 seconds and analyzed using a Zeiss EM 109 electron microscope. Three samples of each genotype were analyzed in duplicates. Deep Sequencing of the preS Region A part of the preS region was amplified by nested PCR in 2 rounds (primers 5_f/5_r and 15_f/15_r). PCR was done with the Titanium Taq PCR Kit (Takara Bio Europe, Saint-Germain-en-Laye, France). For detailed PCR conditions please see Supplementary material. Samples were analyzed by Illumina deep sequencing (Seq-It GmbH, Kaiserslautern, Germany) as described [20, 21]. We used a conservative 1% frequency (occurrence rate of the variant in percent of the quasispecies) cutoff for calling variants [20]. RESULTS Specific Pattern of Large, Middle, and Small HBsAg in Patient Sera Infected With Different HBV Genotypes Virions and SVPs from sera of patients infected with HBV GTA–GTE were enriched by ultracentrifugation and analyzed for the distribution of the different surface proteins (SHBs, MHBs, LHBs) by western blot using the S-domain–specific monoclonal antibody HB01 (representative samples are shown in Figure 1A; characteristics in Supplementary Table S2). Because LHBs of genotype D harbors an 11-amino acid (aa) deletion, the molecular weight of the LHBs signal was detected at a lower molecular weight in comparison to the other genotypes [3]. As expected, the SHBs fraction (24 and 27 kDa) was the largest in all genotypes (Figure 1A). However, the relative amounts of LHBs/MHBs compared to SHBs were higher in genotypes B and D and lower in A, C, and E (Figure 1B). While more LHBs (42 and 39 kDa) than MHBs (36 and 33 kDa) was found in GTB, more MHBs than LHBs was observed in GTC, GTD, and GTE (Figure 1A and 1B). Slightly more MHBs than LHBs was detected in sera from patients with HBV GTA. Figure 1. View largeDownload slide Distribution of large, middle, and small hepatitis B virus surface proteins (LHBs, MHBs, SHBs) varies in HBV genotypes A to E (GTA–GTE). A, Western blot analysis using an HBV surface antigen (HBsAg)-specific antibody (HB01) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV GTA–GTE after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. LHBs and SHBs occur in unglycosylated (LHBs and SHBs) and glycosylated (gLHBs and gSHBs) forms, whereas MHBs occurs in glycosylated (gMHBs) and double glycosylated (ggMHBs) forms. B, Bar chart showing quantification of the relative amounts of LHBs, MHBs, and SHBs in the western blot (A). C, Western blot analysis using antibody HB01 of serum samples from HBeAg-negative HBV-infected patients at baseline (BL) and immediately before they had to start antiviral therapy (TS). Figure 1. View largeDownload slide Distribution of large, middle, and small hepatitis B virus surface proteins (LHBs, MHBs, SHBs) varies in HBV genotypes A to E (GTA–GTE). A, Western blot analysis using an HBV surface antigen (HBsAg)-specific antibody (HB01) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV GTA–GTE after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. LHBs and SHBs occur in unglycosylated (LHBs and SHBs) and glycosylated (gLHBs and gSHBs) forms, whereas MHBs occurs in glycosylated (gMHBs) and double glycosylated (ggMHBs) forms. B, Bar chart showing quantification of the relative amounts of LHBs, MHBs, and SHBs in the western blot (A). C, Western blot analysis using antibody HB01 of serum samples from HBeAg-negative HBV-infected patients at baseline (BL) and immediately before they had to start antiviral therapy (TS). To investigate whether the SHBs to MHBs to LHBs ratios changed during disease progression, sera of patients who had to start treatment (as recommended by the German HBV guideline [7]) during the follow-up of the study were analyzed. Sera collected at baseline and before start of antiviral treatment were analyzed by western blot using the HB01 antibody. In all samples derived from patients infected with GTA–GTD no changes in the SHBs/MHBs/LHBs ratio were observed from baseline to the start of antiviral treatment (examples of GTA–GTD are shown in Figure 1C). Taken together, a genotype-specific pattern of the SHBs/MHBs/LHBs ratio was observed in HBV GTA–GTE. This pattern does not change in sera of patients who experience HBV reactivation. Densities and Morphology of HBsAg-Containing Particles Vary in Different HBV Genotypes For separation of secreted particles in the different genotypes, sucrose density gradient centrifugation of the serum samples and analyses via western blot, HBsAg ELISA, qPCR, and electron microscopy were performed. Due to a low viral replication in our HBeAg-negative HBV-infected patients, very low HBV DNA levels were detected in the qPCR in all fractions (data not shown). The largest HBsAg amounts were found by specific HBsAg-ELISA in fractions 8 and 9 of the GTA, GTC, and GTE samples, whereas the HBsAg peak shifted to fractions with a higher sucrose density (11 and 12) in the GTB and GTD samples (Figure 2A). This phenomenon was also observed in the western blot using the HB01 antibody (Figure 2B). While larger amounts of HBsAg were detected in fractions of a lower density in GTA and GTE samples (fractions 8–10), the HBsAg peak shifted to fractions with a higher density in GTC (fractions 9–12). An even more pronounced shift was detected in GTB and GTD (fractions 10–14). In patients infected with HBV GTB, detectable amounts of LHBs were found in fractions with the highest density (fractions 17 and 18), which was different from the pattern observed in other genotypes. To explore whether HBsAg patterns change during the course of disease, samples of patients (infected with GTA, GTB, GTD, and GTE) who had to start treatment during the follow-up of the study were analyzed. No changes in the density of the secreted particles were observed in patient samples collected before treatment start in comparison to baseline (Figure 2C). Figure 2. View largeDownload slide View largeDownload slide Densities and composition of hepatitis B virus (HBV) surface antigen (HBsAg)-positive particles vary in different HBV genotypes. A, Analysis of sucrose fractions 1–18 of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV genotypes A to E (GTA–GTE) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient (25%–40% sucrose from top to bottom) by HBsAg enzyme-linked immunosorbent assay (ELISA) and refractometry. B, Western blot analysis using HBsAg-specific antibody (HB01) of fractions 1–18 described in (A). C, Western blot analysis using antibody HB01 of serum samples from a HBeAg-negative patients infected with GTD at baseline (BL) and immediately before treatment start (TS) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient. D, Electron microscopy analysis of viral and subviral particles of serum samples from HBeAg-negative patients infected with GTA–GTE after enrichment via ultracentrifugation over a 10% sucrose cushion. Abbreviations: g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; SHBs, small HBV surface protein. Figure 2. View largeDownload slide View largeDownload slide Densities and composition of hepatitis B virus (HBV) surface antigen (HBsAg)-positive particles vary in different HBV genotypes. A, Analysis of sucrose fractions 1–18 of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV genotypes A to E (GTA–GTE) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient (25%–40% sucrose from top to bottom) by HBsAg enzyme-linked immunosorbent assay (ELISA) and refractometry. B, Western blot analysis using HBsAg-specific antibody (HB01) of fractions 1–18 described in (A). C, Western blot analysis using antibody HB01 of serum samples from a HBeAg-negative patients infected with GTD at baseline (BL) and immediately before treatment start (TS) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient. D, Electron microscopy analysis of viral and subviral particles of serum samples from HBeAg-negative patients infected with GTA–GTE after enrichment via ultracentrifugation over a 10% sucrose cushion. Abbreviations: g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; SHBs, small HBV surface protein. Electron microscopy analyses of enriched sera were performed to investigate whether the observed genotype-specific pattern was reflected in a genotype-specific morphology of the secreted particles (Figure 2D). While mostly spheres (diameter 22 nm) and few filaments were observed in GTA, GTC, and GTE, fewer spheres were found in GTB and GTD. Instead, a higher amount of filaments with variable length were found in patients with HBV GTB and GTD. In addition, accumulations of longer filaments to large bundles were observed in GTB. However, only a few Dane particles were found overall, most likely due to the low viral replication in our patients. In summary, secreted HBsAg-containing particles displayed a higher density in HBV GTB and GTD in comparison to GTA, GTC, and GTE. A higher filaments/spheres ratio was observed in HBV GTB and GTD. Mutations in preS1 and preS2 Are Not Directly Reflected by HBsAg Composition In Vivo but In Vitro To analyze the impact of different preS mutations on the HBsAg pattern in these patients’ sera, variants harboring different mutations in the preS1 and/or preS2 domain, as revealed by population-based sequencing [14], were analyzed by western blot using the S-domain–specific antibody HB01, the preS1-specific antibody MA18/7, and the preS2-specific antibody Q19/10. Although, harboring mutations and deletions in the preS domain, which should theoretically affect the molecular weight of the different surface proteins or their expression, no differences were observed in the HBsAg pattern when compared to variants without mutations or deletions. For example, the detected molecular weights for LHBs (39 kDa and 42 kDa) of a GTA variant with a 15-aa deletion in preS1 and a GTA variant with a 49-aa deletion and an additional premature stop appear with a strong signal at the same molecular weight in comparison to LHBs of a GTA variant harboring no mutations (Figure 3A left panel ; for overview of the various mutations/deletion in our patients see Figure 3B). Also, expression signals of LHBs and MHBs (36 and 33 kDa) of a GTD variant harboring a mutation that abolishes the preS2 start codon and a GTD variant harboring 2 deletions in preS2 (5 aa and 3 aa deleted) were detectable and appeared at the same molecular weight in comparison to LHBs and MHBs of a GTD variant without mutations (Figure 3A middle panel). As another example, LHBs and MHBs of a GTC variant harboring a 3-aa deletion in preS2 and a GTC variant harboring an 11-aa deletion in preS2 in combination with a preS2 start codon mutation could be detected and appeared at the same molecular weight in comparison to a GTC variant without mutations (Figure 3A right panel). However, in light of the small difference in the molecular weight due to a deletion of 3 aa, it is likely that this difference was not resolved by the SDS-PAGE. To investigate if the variability in the viral quasispecies might explain the unexpected HBsAg pattern in the western blot, deep sequencing analysis of the 6 patients with preS1 and preS2 mutations (described above) was performed. Thus, preS amplicons were subjected to Illumina deep sequencing analysis. In 5 out of 6 patients, the predominant mutant variant found by direct sequencing was also detected as the major variant (≥69.9% of the quasispecies) in our deep-sequencing analysis (Table 1). In patients number 1 and 6 the variant was even found with a percentage of >99%. Table 1. Variants Found by Population-Based Sequencing and Deep Sequencing Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 aNone other than the stated variant was found by population-based sequencing. Abbreviations: ∆ deletion; qHBsAg, quantitative hepatitis B virus surface antigen. View Large Table 1. Variants Found by Population-Based Sequencing and Deep Sequencing Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 aNone other than the stated variant was found by population-based sequencing. Abbreviations: ∆ deletion; qHBsAg, quantitative hepatitis B virus surface antigen. View Large Figure 3. View largeDownload slide Large deletions and mutations in the preS1 and/or preS2 domain do not affect the appearance of MHBs and LHBs isolated from patients. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01), preS1-specific antibody (MA18/7), and/or preS2-specific antibody (Q19/10) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. B, Comparison of the various mutations/deletions found within the preS domain of the patient variants 1–6 shown in (A). Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; GTA–GTE, HBV genotypes A to E; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; mut., mutation; ref., reference; SHBs, small HBV surface protein. Figure 3. View largeDownload slide Large deletions and mutations in the preS1 and/or preS2 domain do not affect the appearance of MHBs and LHBs isolated from patients. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01), preS1-specific antibody (MA18/7), and/or preS2-specific antibody (Q19/10) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. B, Comparison of the various mutations/deletions found within the preS domain of the patient variants 1–6 shown in (A). Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; GTA–GTE, HBV genotypes A to E; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; mut., mutation; ref., reference; SHBs, small HBV surface protein. To prove that such mutations could affect the HBsAg pattern in western blot analysis, a GTA 1.5-fold HBV genome with the 15-aa deletion in the preS1 domain (termed preS-del.-variant, derived from patient number 1, Figure 3A left panel) was analyzed in vitro in hepatoma cells in comparison to a GTA 1.5-fold genome from a patient without mutations in the preS domain (termed reference genome). Indeed, a shift of the LHBs signal to a lower molecular weight in the preS-del.-variant in comparison to the reference genome was observed in the western blot of lysates of transiently transfected Huh 7.5 cells (Figure 4A). As the specific binding site for the LHBs-specific antibody MA18/7 (D31-F34 of the preS-region [22]) is deleted in the preS-del.-variant, LHBs was not detectable in the western blot analysis of lysates of transiently transfected Huh 7.5 cells (Figure 4B). In contrast, a strong specific LHBs signal was also detected in the serum of the patient with the 15-aa deletion in preS1 and appeared also at the same molecular weight in comparison to the western blot pattern of the patient infected with the reference genome (Figure 4C). Figure 4. View largeDownload slide Expression and molecular weight of LHBs are affected in cells transfected with a preS1 deletion variant. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01) of lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant. Western blot analysis using LHBs-specific antibody (MA18/7) of (B) lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant and (C) serum samples from patients with the ref. genome variant and the preS-del. variant after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; ref., reference; SHBs, small HBV surface protein. Figure 4. View largeDownload slide Expression and molecular weight of LHBs are affected in cells transfected with a preS1 deletion variant. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01) of lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant. Western blot analysis using LHBs-specific antibody (MA18/7) of (B) lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant and (C) serum samples from patients with the ref. genome variant and the preS-del. variant after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; ref., reference; SHBs, small HBV surface protein. Taken together, our in vivo and in vitro data indicate that in HBeAg-negative HBV-infected patients with mutations/deletions in the preS1 and preS2 domain, MHBs and LHBs are expressed from a source that is genetically different from the released viral genomes. DISCUSSION In our study we observed that the composition of secreted HBsAg and the morphology of secreted subviral particles is strongly HBV genotype-dependent in HBeAg-negative chronic HBV-infected patients. We observed that the relative amounts of LHBs/MHBs compared to SHBs were higher in genotypes B and D and lower in A, C, and E, which is in line with another in vivo study in which significant higher proportions of LHBs and MHBs were found in GTD compared to GTA, in patients with chronic hepatitis B [23]. While in our study higher amounts of LHBs than MHBs were found in GTB, higher amounts of MHBs than LHBs were observed in GTC, GTD, and GTE, which is partly in contrast to 2 recently published in vitro studies describing generally higher amounts of LHBs than MHBs in genotypes A–D and J [16, 24]. On the one hand, differences in the in vitro data might be due to the individual genomes that were taken to be representative of the different genotypes in the in vitro studies. On the other hand, HBeAg-positive HBV genomes were analyzed in the in vitro studies, whereas sera of HBeAg-negative chronic HBV-infected patients were analyzed in our study. Recently, lower LHBs and MHBs amounts were reported to be characteristic of inactive carriers compared to patients in other disease stages [23]. Therefore, genotype-specific differences in HBsAg composition might also depend on the disease stage (ie, HBeAg positive vs HBeAg negative). In addition, as filaments are characterized by a higher content of LHBs than spheres, we found that differences in HBsAg composition reflected variations in the density and morphology of secreted SVPs [3]. While Hassemer et al. could not detect any differences regarding density of HBsAg-containing particles among genotypes A–D in vitro, we found that HBsAg-containing particles in GTB and GTD-infected patients displayed a higher density [16]. Furthermore, electron microscopy analyses in our study indicated an increased ratio of filaments to spheres in sera of GTB and GTD, and long filaments accumulating to larger bundles in sera of GTB-infected patients. As SVPs represent the predominant particle type in HBV-infected patients and as they are essential for the persistence of the infection [6, 25], genotype-specific differences in the morphology and ratio of different SVPs may result in a genotype-specific immunogenicity and pathogenesis. By comparing different preS1 and preS2 deletions and mutations present in HBV genomes of circulating virions with the structure of SVPs in the circulation of the same patients, we saw no detectable effect of the observed genetic changes on the composition or size of the HBs proteins in the SVPs. Neither deletions in preS1 nor in preS2 led to the formation of LHBs or MHBs with a lower molecular weight. Moreover, mutations abolishing the preS2 start codon had no impact on MHBs expression. We excluded variability in the viral quasispecies as an explanation for the unexpected MHBs and LHBs pattern from deep sequencing analyses, as these mutations were mainly the major variants of the quasispecies. However, in vitro analyses of a GTA genome harboring a large preS1 deletion proved that this deletion results in a truncated form of LHBs as reflected by a decreased molecular weight when expressed in vitro in comparison to our observation in the patient infected with this variant. In addition, a strong specific LHBs signal was detected in a serum sample from this patient using a preS1-specific antibody, although the preS1 deletion abolishes the binding site of this antibody as observed in vitro. Therefore, in the analyzed patients harboring preS1/preS2 mutations, LHBs and MHBs must be expressed from a genetic source that is different from the secreted viral genomes in which these mutations were detected. The nuclear cccDNA has been reported to serve as a template for the synthesis of the pregenomic RNA, which is an intermediate for viral replication and virion assembly [2]. Therefore, secreted HBV DNA is derived from cccDNA. Also, in HBe-negative patients serum HBV DNA was observed to reflect quantitative amounts of cccDNA [26]. Thus, while the viral genomes analyzed in our study were derived from cccDNA, MHBs and LHBs must be derived from a different source, which is most likely the integrated DNA. This is in line with 2 studies that showed no correlation between cccDNA amounts and HBsAg serum levels in HBeAg-negative patients [26, 27], while in HBeAg-positive patients a strong correlation was found [27]. Both, our data and these studies suggest that HBV envelope proteins can be expressed in adequate amounts also from the integrated DNA in HBeAg-negative chronic HBV-infected patients. If HBsAg from integrated HBV genomes also transcomplements viral particle synthesis in addition to the formation of SVPs is uncertain. One potential hypothesis could be that transcomplementation occurs by these intact surface proteins from integrated HBV genomes enveloping nucleocapsids that harbor the defective HBV genome derived from cccDNA. However, as absence of MHBs and certain LHBs deletions can be tolerated for virion production, virions with mutated MHBs and LHBs may exist together with wild-type SVPs expressed by other cells. But if replication-active cccDNA coexists with HBsAg expressing integrated HBV DNA within one cell, a transcomplementation of intact HBsAg in the envelope of the virions could occur. HBsAg expression from integrated DNA was also assumed in a recent trial exploring an siRNA targeting the cccDNA in HBV-infected patients. In this trial, HBsAg was not sufficiently reduced by treatment with the siRNA in HBeAg-negative patients, while a sufficient reduction was observed in HBeAg-positive chronically HBV-infected patients [28]. The authors concluded that HBsAg was derived from integrated DNA instead of cccDNA and was therefore not sufficiently reduced by the cccDNA-specific siRNA. However, loss of HBsAg, which is also regarded as a functional cure, is generally considered an important treatment goal in patients with chronic HBV infection and several novel treatment concepts also targeting HBsAg secretion are in preclinical and clinical evaluation [13]. Thus, integrated DNA, as a potential and most likely highly productive HBsAg source, has to be considered for new antiviral strategies in HBeAg-negative patients in order to induce HBsAg loss in a higher number of patients. In conclusion, HBsAg composition and morphology of subviral particles differ among HBV genotypes A–E. PreS mutations and deletions do affect HBsAg expression in vitro but no detectable effect of the observed genetic changes on the composition or size of the HBs proteins were found in our patients. Our data strongly suggest that HBsAg can be expressed in adequate amounts also from the integrated DNA in HBeAg-negative chronic HBV-infected patients. This must be considered when developing novel treatment strategies in these patients. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This work was supported by Deutsches Zentrum für Infektionsforschung (Thematic Translational Units Hepatitis) (C. S.); Deutsche Forschungsgemeinschaft (grant numbers PE 2403/2-1 (K. P.) and HI 858/12-1 (E. H.); and a grant from the Ellen Schairer-Stiftung (K. P.). Potential conflicts of interest. J. V. reports consultancies and speaker: Abbott, AbbVie, Gilead, BMS, Medtronic, Merck/MSD, and Roche. C. S. reports consultancies and speaker: Abbvie, Gilead, BMS, Intercept, Janssen, and Merck/MSD; and research support: Gilead. S. Z. reports consultancies: Abbvie, BMS, Gilead, Intercept, Janssen, and Merck/MSD. All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. World Health Organization . Hepatitis B fact sheet, updated April 2017 . http://www.who.int/mediacentre/factsheets/fs204/en/. Accessed 16 March 2018 . 2. Schädler S , Hildt E . HBV life cycle: entry and morphogenesis . Viruses 2009 ; 1 : 185 – 209 . Google Scholar CrossRef Search ADS PubMed 3. Heermann KH , Goldmann U , Schwartz W , Seyffarth T , Baumgarten H , Gerlich WH . Large surface proteins of hepatitis B virus containing the pre-s sequence . J Virol 1984 ; 52 : 396 – 402 . Google Scholar PubMed 4. Patient R , Hourioux C , Roingeard P . Morphogenesis of hepatitis B virus and its subviral envelope particles . Cell Microbiol 2009 ; 11 : 1561 – 70 . Google Scholar CrossRef Search ADS PubMed 5. Jiang B , Himmelsbach K , Ren H , Boller K , Hildt E . Subviral hepatitis B virus filaments, like infectious viral particles, are released via multivesicular bodies . J Virol 2015 ; 90 : 3330 – 41 . Google Scholar CrossRef Search ADS PubMed 6. Cornberg M , Wong VW , Locarnini S , Brunetto M , Janssen HLA , Chan HL . The role of quantitative hepatitis B surface antigen revisited . J Hepatol 2017 ; 66 : 398 – 411 . Google Scholar CrossRef Search ADS PubMed 7. Cornberg M , Protzer U , Petersen J et al. ; AWMF . Prophylaxis, diagnosis and therapy of hepatitis B virus infection - the German guideline . Z Gastroenterol 2011 ; 49 : 871 – 930 . Google Scholar CrossRef Search ADS PubMed 8. European Association for the Study of the Liver . EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection . J Hepatol 2017 ; 67 : 370 – 98 . CrossRef Search ADS PubMed 9. Terrault NA , Bzowej NH , Chang KM , Hwang JP , Jonas MM , Murad MH ; American Association for the Study of Liver Diseases . AASLD guidelines for treatment of chronic hepatitis B . Hepatology 2016 ; 63 : 261 – 83 . Google Scholar CrossRef Search ADS PubMed 10. Sarin SK , Kumar M , Lau GK et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update . Hepatol Int 2016 ; 10 : 1 – 98 . Google Scholar CrossRef Search ADS PubMed 11. Lucifora J , Protzer U . Attacking hepatitis B virus cccDNA–the holy grail to hepatitis B cure . J Hepatol 2016 ; 64 : 41 – 8 . Google Scholar CrossRef Search ADS 12. Tu T , Budzinska MA , Shackel NA , Urban S . HBV DNA integration: molecular mechanisms and clinical implications . Viruses 2017 ; 9 : E75 . Google Scholar CrossRef Search ADS PubMed 13. Durantel D , Zoulim F . New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus . J Hepatol 2016 ; 64 : 117 – 31 . Google Scholar CrossRef Search ADS 14. Kuhnhenn L , Jiang B , Kubesch A , et al. Impact of HBV genotype and mutations on HBV DNA and qHBsAg levels in patients with HBeAg-negative chronic HBV infection. Aliment Pharmacol Ther. 2018;1–13 . 15. Riveiro-Barciela M , Bes M , Rodríguez-Frías F et al. Serum hepatitis B core-related antigen is more accurate than hepatitis B surface antigen to identify inactive carriers, regardless of hepatitis B virus genotype . Clin Microbiol Infect 2017 ; 23 : 860 – 7 . Google Scholar CrossRef Search ADS PubMed 16. Hassemer M , Finkernagel M , Peiffer KH et al. Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes . Virology 2017 ; 502 : 1 – 12 . Google Scholar CrossRef Search ADS PubMed 17. Ausubel F , Brent R , Kingston R , Moore D , Seidman J , Smith J. Current protocols in molecular biology . In: Gallagher SR (unit 10.2); Sasse J, Gallagher SR (unit 10.7); Gallagher S, Winston SE, Fuller SA, Hurrell JGR (unit 10.8), eds. John Wiley , 2003 ; Supplement 66. 18. Kucinskaite-Kodze I , Pleckaityte M , Bremer CM et al. New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen . Virus Res 2016 ; 211 : 209 – 21 . Google Scholar CrossRef Search ADS PubMed 19. Deepen R , Heermann KH , Uy A , Thomssen R , Gerlich WH . Assay of preS epitopes and preS1 antibody in hepatitis B virus carriers and immune persons . Med Microbiol Immunol 1990 ; 179 : 49 – 60 . Google Scholar CrossRef Search ADS PubMed 20. Dietz J , Schelhorn SE , Fitting D et al. Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients . J Virol 2013 ; 87 : 6172 – 81 . Google Scholar CrossRef Search ADS PubMed 21. Susser S , Flinders M , Reesink HW et al. Evolution of hepatitis C virus quasispecies during repeated treatment with the NS3/4A protease inhibitor telaprevir . Antimicrob Agents Chemother 2015 ; 59 : 2746 – 55 . Google Scholar CrossRef Search ADS PubMed 22. Sominskaya I , Pushko P , Dreilina D , Kozlovskaya T , Pumpen P . Determination of the minimal length of preS1 epitope recognized by a monoclonal antibody which inhibits attachment of hepatitis B virus to hepatocytes . Med Microbiol Immunol 1992 ; 181 : 215 – 26 . Google Scholar CrossRef Search ADS PubMed 23. Pfefferkorn M , Bohm S , Schott T et al. Quantification of large and middle proteins of hepatitis B virus surface antigen (HBsAg) as a novel tool for the identification of inactive HBV carriers . Gut [published online ahead of print 26 September, 2017]. doi: 10.1136/gutjnl-2017-313811 . 24. Sozzi V , Walsh R , Littlejohn M et al. In vitro studies show that sequence variability contributes to marked variation in hepatitis B virus replication, protein expression, and function observed across genotypes . J Virol 2016 ; 90 : 10054 – 64 . Google Scholar CrossRef Search ADS PubMed 25. Rydell GE , Prakash K , Norder H , Lindh M . Hepatitis B surface antigen on subviral particles reduces the neutralizing effect of anti-HBs antibodies on hepatitis B viral particles in vitro . Virology 2017 ; 509 : 67 – 70 . Google Scholar CrossRef Search ADS PubMed 26. Lin LY , Wong VW , Zhou HJ et al. Relationship between serum hepatitis B virus DNA and surface antigen with covalently closed circular DNA in HBeAg-negative patients . J Med Virol 2010 ; 82 : 1494 – 500 . Google Scholar CrossRef Search ADS PubMed 27. Thompson AJ , Nguyen T , Iser D et al. Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers . Hepatology 2010 ; 51 : 1933 – 44 . Google Scholar CrossRef Search ADS PubMed 28. Wooddell CI , Yuen MF , Chan HL et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg . Sci Transl Med 2017 ; 9 : eaan0241 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Divergent preS Sequences in Virion-Associated Hepatitis B Virus Genomes and Subviral HBV Surface Antigen Particles From HBV e Antigen-Negative Patients

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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1537-6613
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10.1093/infdis/jiy119
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Abstract

Abstract Background Hepatitis B virus (HBV) surface proteins (HBsAg) coat the viral particle and form subviral particles (SVPs). Loss of HBsAg represents a functional cure and is an important treatment goal. Methods We analyzed the impact of the HBV genotypes A–E and pre-S mutations on SVP expression in hepatitis B virus e antigen (HBeAg)-negative chronic HBV-infected patients. A HBV genome harboring a preS1-deletion was analyzed in hepatoma cells. Results We observed a genotype-specific ratio of the 3 surface proteins (SHBs/MHBs/LHBs), reflecting differences in the morphology and composition of SVPs. Deletions/mutations in the preS1/preS2 domain, detected in released viral genomes, did not affect the molecular weight of MHBs and LHBs in these patients. In contrast, LHB molecular weight was altered in vitro using an HBV genome harboring a preS1-deletion derived from one of these patients. Conclusion Differences in composition of SVPs may result in genotype-specific immunogenicity and pathogenesis. In the patients with preS-mutations, secreted HBsAg and released viral genomes cannot be derived from the same genetic source. As viral genomes are derived from covalently closed circular DNA (cccDNA), HBsAg is presumably derived from integrated DNA. This important HBsAg source should be considered for novel antiviral strategies in HBeAg-negative chronic HBV-infected patients. HBV, cccDNA, integrated DNA, HBsAg, filaments, spheres Despite the availability of an effective vaccine, chronic infection with the hepatitis B virus (HBV) is still a major cause of severe liver disease [1]. Its circular 3.2-kb genome contains 4 open reading frames, which encode the preC/Core protein, the regulatory X protein, the viral polymerase, and surface proteins [2]. The 3 surface proteins are encoded by 1 open reading frame containing 3 in-frame AUG start codons. Depending on the initiating site, large surface protein (LHBs; preS1, preS2, and s domain), middle surface protein (MHBs; preS2 and s), or small surface protein (SHBs; only s) are expressed [3]. The surface protein (HBsAg) coats the infectious viral particle (Dane particle). In addition, subviral particles (SVPs) are highly overproduced from HBV infected cells. These particles consist only of HBsAg and lipids and are divided according to their shape into filaments of variable length and 22-nm spheres. While spheres are released via the general secretory pathway, filaments like infectious viral particles are released via so-called multivesicular bodies [4, 5]. However, the role of SVPs in HBV-related pathogenesis is not fully understood. Although they are noninfectious, they have been reported to distract neutralizing antibodies from the virus, thereby allowing viral spread and persistence in the host [6]. Either nucleot(s)ide analogues (NA) or pegylated interferon, depending on viral load, presence of liver inflammation, and/or liver fibrosis, are recommended for antiviral treatment for HBV-related hepatitis [7–10]. While sufficient suppression of viral replication can be achieved with NA treatment, HBsAg loss as a functional cure is a rare event due to the presence of covalently closed circular DNA (cccDNA) and integrated DNA. The cccDNA is organized into a chromatin-like structure and can persist for decades in the human liver [11]. Integration of HBV DNA can occur through the entire host’s genome. However, so far no distinct integration sites have been identified in nontumor tissues [12]. Transcription of HBsAg from integrated DNA is still possible as the HBsAg open reading frame remains intact in most cases and retains its 2 native promoters [12]. Several new antiviral concepts targeting different sites of the viral life cycle, including HBsAg release, are currently in development [13]. Recently, it was reported that HBsAg serum levels are associated with HBV genotypes and frequent mutations in precore and preS in patients with HBV e antigen (HBeAg)-negative HBV infection [14, 15]. In the present study we extend our recent in vitro analysis [16] of HBV genotypes by studying the impact of HBV genotypes and mutations in the preS domain on the composition and morphology of SVPs in patients from a large European cohort of HBeAg-negative HBV-infected patients. MATERIALS AND METHODS Patient Samples Serum samples from HBeAg-negative patients (HBV DNA <100000 IU/mL, alanine transaminase <2 × upper limit of normal) infected with human HBV genotype A–E (GTA–GTE) from the German multicenter Albatros trial (listed at Clinical.Trial.gov: NCT01090531) were enrolled for analysis. The Albatros study is an observational long-term follow-up study of patients with HBeAg-negative HBV infection with low-level viremia who do not require antiviral therapy. Main inclusion and exclusion criteria are listed in the Supplementary material. Serum from these patients was collected and stored at −80°C. Quantitative serum HBsAg was measured with the Abbott Architect platform (Abbott Diagnostics) during clinical routine. The study was approved by local ethics committees and written informed consent was obtained from all patients. The study was performed in accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines. Sucrose Density Gradient Centrifugation Viral and subviral particles from patient sera were either enriched or separated via ultracentrifugation. For enrichment, 100–200 µL patient sera were layered on top of a cushion of 10% (w/v) d(+) sucrose dissolved in TN150 buffer and centrifuged in a SW60 rotor (Beckman Coulter, Krefeld, Germany) at 42000 rpm for 2.5 hours at 4°C; 40 µL of TN150 buffer was used for resuspension. For separation, 100–200 µL patient sera (GTA n = 9; GTB n = 7; GTC n = 4; GTD n = 12; GTE n = 2) were layered onto a linear gradient and centrifuged in an SW60 rotor at 32000 rpm for 18 hours at 4°C. The gradient was composed from bottom to top as follows: 40%, 37.5%, 35%, 32,5%, 30%, 27.5%, 25% (w/v) d(+) sucrose dissolved in TN150 buffer. SDS-PAGE and Western Blot Analysis Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were performed according to standard procedures [17]. The primary antibodies anti-β-actin (mouse, monoclonal, Sigma-Aldrich, Seelze, Germany), anti-transferrin (rabbit, monoclonal, Santa Cruz Biotechnology, Heidelberg, Germany), anti-SHBs (HB01, mouse, monoclonal, kindly provided by Aurelia Zvirbliene, Lithuania) [18], anti-LHBs (MA18/7, mouse, monoclonal, kindly provided by Dieter Glebe, Gießen) [3], and anti-preS2 (Q19/10, mouse, monoclonal, kindly provided by Dieter Glebe, Gießen) [19] were used. Peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). At least 10 samples of each genotype were enriched and equal protein amounts, determined via Bradford assay, were analyzed via western blot analysis. Plasmids HBV genomes of 1.5-fold length, derived from 2 HBeAg-negative HBV GTA-infected patients were used: (1) preS1 deletion of 45 nucleotides at positions 2944–2988; and (2) no mutation or deletion regarding the preS1/preS2 domain [14]. Transient Transfection Huh7.5 cells were transiently transfected using 1 µg DNA and 6 µL linear polyethyleneimine (Polysciences Europe GmbH, Hirschberg, Germany) and grown for 48 or 72 hours. HBsAg ELISA HBsAg quantities were analyzed in fractions of sucrose density gradient centrifugation using the Enzygnost HBsAg 6.0 ELISA (Siemens HealthCare Diagnostics, Erlangen, Germany) according to the manufacturer’s instructions and were specified as signal to cutoff ratios. Real-Time PCR Quantitative real time polymerase chain reaction (qPCR) was performed using a custom-designed TaqMan gene expression assay (Thermo Fisher Scientific, Darmstadt, Germany), described in the Supplementary material. Primers Primers used in this study are listed in Supplementary Table S1. Electron Microscopy Samples were incubated with carbon-coated nickel grids for 3 minutes followed by fixation with formaldehyde. Washed grids were negatively stained with a 2% aqueous uranyl acetate solution for 10 seconds and analyzed using a Zeiss EM 109 electron microscope. Three samples of each genotype were analyzed in duplicates. Deep Sequencing of the preS Region A part of the preS region was amplified by nested PCR in 2 rounds (primers 5_f/5_r and 15_f/15_r). PCR was done with the Titanium Taq PCR Kit (Takara Bio Europe, Saint-Germain-en-Laye, France). For detailed PCR conditions please see Supplementary material. Samples were analyzed by Illumina deep sequencing (Seq-It GmbH, Kaiserslautern, Germany) as described [20, 21]. We used a conservative 1% frequency (occurrence rate of the variant in percent of the quasispecies) cutoff for calling variants [20]. RESULTS Specific Pattern of Large, Middle, and Small HBsAg in Patient Sera Infected With Different HBV Genotypes Virions and SVPs from sera of patients infected with HBV GTA–GTE were enriched by ultracentrifugation and analyzed for the distribution of the different surface proteins (SHBs, MHBs, LHBs) by western blot using the S-domain–specific monoclonal antibody HB01 (representative samples are shown in Figure 1A; characteristics in Supplementary Table S2). Because LHBs of genotype D harbors an 11-amino acid (aa) deletion, the molecular weight of the LHBs signal was detected at a lower molecular weight in comparison to the other genotypes [3]. As expected, the SHBs fraction (24 and 27 kDa) was the largest in all genotypes (Figure 1A). However, the relative amounts of LHBs/MHBs compared to SHBs were higher in genotypes B and D and lower in A, C, and E (Figure 1B). While more LHBs (42 and 39 kDa) than MHBs (36 and 33 kDa) was found in GTB, more MHBs than LHBs was observed in GTC, GTD, and GTE (Figure 1A and 1B). Slightly more MHBs than LHBs was detected in sera from patients with HBV GTA. Figure 1. View largeDownload slide Distribution of large, middle, and small hepatitis B virus surface proteins (LHBs, MHBs, SHBs) varies in HBV genotypes A to E (GTA–GTE). A, Western blot analysis using an HBV surface antigen (HBsAg)-specific antibody (HB01) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV GTA–GTE after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. LHBs and SHBs occur in unglycosylated (LHBs and SHBs) and glycosylated (gLHBs and gSHBs) forms, whereas MHBs occurs in glycosylated (gMHBs) and double glycosylated (ggMHBs) forms. B, Bar chart showing quantification of the relative amounts of LHBs, MHBs, and SHBs in the western blot (A). C, Western blot analysis using antibody HB01 of serum samples from HBeAg-negative HBV-infected patients at baseline (BL) and immediately before they had to start antiviral therapy (TS). Figure 1. View largeDownload slide Distribution of large, middle, and small hepatitis B virus surface proteins (LHBs, MHBs, SHBs) varies in HBV genotypes A to E (GTA–GTE). A, Western blot analysis using an HBV surface antigen (HBsAg)-specific antibody (HB01) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV GTA–GTE after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. LHBs and SHBs occur in unglycosylated (LHBs and SHBs) and glycosylated (gLHBs and gSHBs) forms, whereas MHBs occurs in glycosylated (gMHBs) and double glycosylated (ggMHBs) forms. B, Bar chart showing quantification of the relative amounts of LHBs, MHBs, and SHBs in the western blot (A). C, Western blot analysis using antibody HB01 of serum samples from HBeAg-negative HBV-infected patients at baseline (BL) and immediately before they had to start antiviral therapy (TS). To investigate whether the SHBs to MHBs to LHBs ratios changed during disease progression, sera of patients who had to start treatment (as recommended by the German HBV guideline [7]) during the follow-up of the study were analyzed. Sera collected at baseline and before start of antiviral treatment were analyzed by western blot using the HB01 antibody. In all samples derived from patients infected with GTA–GTD no changes in the SHBs/MHBs/LHBs ratio were observed from baseline to the start of antiviral treatment (examples of GTA–GTD are shown in Figure 1C). Taken together, a genotype-specific pattern of the SHBs/MHBs/LHBs ratio was observed in HBV GTA–GTE. This pattern does not change in sera of patients who experience HBV reactivation. Densities and Morphology of HBsAg-Containing Particles Vary in Different HBV Genotypes For separation of secreted particles in the different genotypes, sucrose density gradient centrifugation of the serum samples and analyses via western blot, HBsAg ELISA, qPCR, and electron microscopy were performed. Due to a low viral replication in our HBeAg-negative HBV-infected patients, very low HBV DNA levels were detected in the qPCR in all fractions (data not shown). The largest HBsAg amounts were found by specific HBsAg-ELISA in fractions 8 and 9 of the GTA, GTC, and GTE samples, whereas the HBsAg peak shifted to fractions with a higher sucrose density (11 and 12) in the GTB and GTD samples (Figure 2A). This phenomenon was also observed in the western blot using the HB01 antibody (Figure 2B). While larger amounts of HBsAg were detected in fractions of a lower density in GTA and GTE samples (fractions 8–10), the HBsAg peak shifted to fractions with a higher density in GTC (fractions 9–12). An even more pronounced shift was detected in GTB and GTD (fractions 10–14). In patients infected with HBV GTB, detectable amounts of LHBs were found in fractions with the highest density (fractions 17 and 18), which was different from the pattern observed in other genotypes. To explore whether HBsAg patterns change during the course of disease, samples of patients (infected with GTA, GTB, GTD, and GTE) who had to start treatment during the follow-up of the study were analyzed. No changes in the density of the secreted particles were observed in patient samples collected before treatment start in comparison to baseline (Figure 2C). Figure 2. View largeDownload slide View largeDownload slide Densities and composition of hepatitis B virus (HBV) surface antigen (HBsAg)-positive particles vary in different HBV genotypes. A, Analysis of sucrose fractions 1–18 of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV genotypes A to E (GTA–GTE) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient (25%–40% sucrose from top to bottom) by HBsAg enzyme-linked immunosorbent assay (ELISA) and refractometry. B, Western blot analysis using HBsAg-specific antibody (HB01) of fractions 1–18 described in (A). C, Western blot analysis using antibody HB01 of serum samples from a HBeAg-negative patients infected with GTD at baseline (BL) and immediately before treatment start (TS) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient. D, Electron microscopy analysis of viral and subviral particles of serum samples from HBeAg-negative patients infected with GTA–GTE after enrichment via ultracentrifugation over a 10% sucrose cushion. Abbreviations: g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; SHBs, small HBV surface protein. Figure 2. View largeDownload slide View largeDownload slide Densities and composition of hepatitis B virus (HBV) surface antigen (HBsAg)-positive particles vary in different HBV genotypes. A, Analysis of sucrose fractions 1–18 of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV genotypes A to E (GTA–GTE) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient (25%–40% sucrose from top to bottom) by HBsAg enzyme-linked immunosorbent assay (ELISA) and refractometry. B, Western blot analysis using HBsAg-specific antibody (HB01) of fractions 1–18 described in (A). C, Western blot analysis using antibody HB01 of serum samples from a HBeAg-negative patients infected with GTD at baseline (BL) and immediately before treatment start (TS) after separation of subviral particles via ultracentrifugation over a continuous sucrose gradient. D, Electron microscopy analysis of viral and subviral particles of serum samples from HBeAg-negative patients infected with GTA–GTE after enrichment via ultracentrifugation over a 10% sucrose cushion. Abbreviations: g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; SHBs, small HBV surface protein. Electron microscopy analyses of enriched sera were performed to investigate whether the observed genotype-specific pattern was reflected in a genotype-specific morphology of the secreted particles (Figure 2D). While mostly spheres (diameter 22 nm) and few filaments were observed in GTA, GTC, and GTE, fewer spheres were found in GTB and GTD. Instead, a higher amount of filaments with variable length were found in patients with HBV GTB and GTD. In addition, accumulations of longer filaments to large bundles were observed in GTB. However, only a few Dane particles were found overall, most likely due to the low viral replication in our patients. In summary, secreted HBsAg-containing particles displayed a higher density in HBV GTB and GTD in comparison to GTA, GTC, and GTE. A higher filaments/spheres ratio was observed in HBV GTB and GTD. Mutations in preS1 and preS2 Are Not Directly Reflected by HBsAg Composition In Vivo but In Vitro To analyze the impact of different preS mutations on the HBsAg pattern in these patients’ sera, variants harboring different mutations in the preS1 and/or preS2 domain, as revealed by population-based sequencing [14], were analyzed by western blot using the S-domain–specific antibody HB01, the preS1-specific antibody MA18/7, and the preS2-specific antibody Q19/10. Although, harboring mutations and deletions in the preS domain, which should theoretically affect the molecular weight of the different surface proteins or their expression, no differences were observed in the HBsAg pattern when compared to variants without mutations or deletions. For example, the detected molecular weights for LHBs (39 kDa and 42 kDa) of a GTA variant with a 15-aa deletion in preS1 and a GTA variant with a 49-aa deletion and an additional premature stop appear with a strong signal at the same molecular weight in comparison to LHBs of a GTA variant harboring no mutations (Figure 3A left panel ; for overview of the various mutations/deletion in our patients see Figure 3B). Also, expression signals of LHBs and MHBs (36 and 33 kDa) of a GTD variant harboring a mutation that abolishes the preS2 start codon and a GTD variant harboring 2 deletions in preS2 (5 aa and 3 aa deleted) were detectable and appeared at the same molecular weight in comparison to LHBs and MHBs of a GTD variant without mutations (Figure 3A middle panel). As another example, LHBs and MHBs of a GTC variant harboring a 3-aa deletion in preS2 and a GTC variant harboring an 11-aa deletion in preS2 in combination with a preS2 start codon mutation could be detected and appeared at the same molecular weight in comparison to a GTC variant without mutations (Figure 3A right panel). However, in light of the small difference in the molecular weight due to a deletion of 3 aa, it is likely that this difference was not resolved by the SDS-PAGE. To investigate if the variability in the viral quasispecies might explain the unexpected HBsAg pattern in the western blot, deep sequencing analysis of the 6 patients with preS1 and preS2 mutations (described above) was performed. Thus, preS amplicons were subjected to Illumina deep sequencing analysis. In 5 out of 6 patients, the predominant mutant variant found by direct sequencing was also detected as the major variant (≥69.9% of the quasispecies) in our deep-sequencing analysis (Table 1). In patients number 1 and 6 the variant was even found with a percentage of >99%. Table 1. Variants Found by Population-Based Sequencing and Deep Sequencing Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 aNone other than the stated variant was found by population-based sequencing. Abbreviations: ∆ deletion; qHBsAg, quantitative hepatitis B virus surface antigen. View Large Table 1. Variants Found by Population-Based Sequencing and Deep Sequencing Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 Sample HBV Genotype qHBsAg (IU/mL) Variant Found by Population-Based Sequencinga Variant Found by Deep Sequencing Percentage of the Viral Quasispecies 1 A 4.5 15 aa ∆ preS1 15 aa ∆ preS1 99.2 2 A 4.4 stop in preS1 + 49 aa ∆ preS1 stop in preS1 +/− 49 aa ∆ preS1 84.7 / 10.9 3 D 4.1 preS2 start codon mutation preS2 start codon mutation 88.7 4 D 4.0 5 aa + 3 aa ∆ preS2 5 aa + 3 aa ∆ preS2 69.9 5 C 3.9 3 aa ∆ preS2 3 aa ∆ preS2 17.4 6 C 3.9 11 aa ∆ preS2 + preS2 start codon mutation 11 aa ∆ preS2 + preS2 start codon mutation 99.9 aNone other than the stated variant was found by population-based sequencing. Abbreviations: ∆ deletion; qHBsAg, quantitative hepatitis B virus surface antigen. View Large Figure 3. View largeDownload slide Large deletions and mutations in the preS1 and/or preS2 domain do not affect the appearance of MHBs and LHBs isolated from patients. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01), preS1-specific antibody (MA18/7), and/or preS2-specific antibody (Q19/10) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. B, Comparison of the various mutations/deletions found within the preS domain of the patient variants 1–6 shown in (A). Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; GTA–GTE, HBV genotypes A to E; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; mut., mutation; ref., reference; SHBs, small HBV surface protein. Figure 3. View largeDownload slide Large deletions and mutations in the preS1 and/or preS2 domain do not affect the appearance of MHBs and LHBs isolated from patients. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01), preS1-specific antibody (MA18/7), and/or preS2-specific antibody (Q19/10) of serum samples from HBV e antigen (HBeAg)-negative patients infected with HBV after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. B, Comparison of the various mutations/deletions found within the preS domain of the patient variants 1–6 shown in (A). Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; GTA–GTE, HBV genotypes A to E; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; mut., mutation; ref., reference; SHBs, small HBV surface protein. To prove that such mutations could affect the HBsAg pattern in western blot analysis, a GTA 1.5-fold HBV genome with the 15-aa deletion in the preS1 domain (termed preS-del.-variant, derived from patient number 1, Figure 3A left panel) was analyzed in vitro in hepatoma cells in comparison to a GTA 1.5-fold genome from a patient without mutations in the preS domain (termed reference genome). Indeed, a shift of the LHBs signal to a lower molecular weight in the preS-del.-variant in comparison to the reference genome was observed in the western blot of lysates of transiently transfected Huh 7.5 cells (Figure 4A). As the specific binding site for the LHBs-specific antibody MA18/7 (D31-F34 of the preS-region [22]) is deleted in the preS-del.-variant, LHBs was not detectable in the western blot analysis of lysates of transiently transfected Huh 7.5 cells (Figure 4B). In contrast, a strong specific LHBs signal was also detected in the serum of the patient with the 15-aa deletion in preS1 and appeared also at the same molecular weight in comparison to the western blot pattern of the patient infected with the reference genome (Figure 4C). Figure 4. View largeDownload slide Expression and molecular weight of LHBs are affected in cells transfected with a preS1 deletion variant. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01) of lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant. Western blot analysis using LHBs-specific antibody (MA18/7) of (B) lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant and (C) serum samples from patients with the ref. genome variant and the preS-del. variant after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; ref., reference; SHBs, small HBV surface protein. Figure 4. View largeDownload slide Expression and molecular weight of LHBs are affected in cells transfected with a preS1 deletion variant. A, Western blot analysis using hepatitis B virus (HBV) surface antigen (HBsAg)-specific antibody (HB01) of lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant. Western blot analysis using LHBs-specific antibody (MA18/7) of (B) lysates of Huh7.5 cells transfected with ref. genome and preS-del.-variant and (C) serum samples from patients with the ref. genome variant and the preS-del. variant after enrichment of viral/subviral particles via ultracentrifugation over a 10% sucrose cushion. Abbreviations: del., deletion; g, glycosylated; gg, double glycosylated; LHBs, large HBV surface protein; MHBs, middle HBV surface protein; ref., reference; SHBs, small HBV surface protein. Taken together, our in vivo and in vitro data indicate that in HBeAg-negative HBV-infected patients with mutations/deletions in the preS1 and preS2 domain, MHBs and LHBs are expressed from a source that is genetically different from the released viral genomes. DISCUSSION In our study we observed that the composition of secreted HBsAg and the morphology of secreted subviral particles is strongly HBV genotype-dependent in HBeAg-negative chronic HBV-infected patients. We observed that the relative amounts of LHBs/MHBs compared to SHBs were higher in genotypes B and D and lower in A, C, and E, which is in line with another in vivo study in which significant higher proportions of LHBs and MHBs were found in GTD compared to GTA, in patients with chronic hepatitis B [23]. While in our study higher amounts of LHBs than MHBs were found in GTB, higher amounts of MHBs than LHBs were observed in GTC, GTD, and GTE, which is partly in contrast to 2 recently published in vitro studies describing generally higher amounts of LHBs than MHBs in genotypes A–D and J [16, 24]. On the one hand, differences in the in vitro data might be due to the individual genomes that were taken to be representative of the different genotypes in the in vitro studies. On the other hand, HBeAg-positive HBV genomes were analyzed in the in vitro studies, whereas sera of HBeAg-negative chronic HBV-infected patients were analyzed in our study. Recently, lower LHBs and MHBs amounts were reported to be characteristic of inactive carriers compared to patients in other disease stages [23]. Therefore, genotype-specific differences in HBsAg composition might also depend on the disease stage (ie, HBeAg positive vs HBeAg negative). In addition, as filaments are characterized by a higher content of LHBs than spheres, we found that differences in HBsAg composition reflected variations in the density and morphology of secreted SVPs [3]. While Hassemer et al. could not detect any differences regarding density of HBsAg-containing particles among genotypes A–D in vitro, we found that HBsAg-containing particles in GTB and GTD-infected patients displayed a higher density [16]. Furthermore, electron microscopy analyses in our study indicated an increased ratio of filaments to spheres in sera of GTB and GTD, and long filaments accumulating to larger bundles in sera of GTB-infected patients. As SVPs represent the predominant particle type in HBV-infected patients and as they are essential for the persistence of the infection [6, 25], genotype-specific differences in the morphology and ratio of different SVPs may result in a genotype-specific immunogenicity and pathogenesis. By comparing different preS1 and preS2 deletions and mutations present in HBV genomes of circulating virions with the structure of SVPs in the circulation of the same patients, we saw no detectable effect of the observed genetic changes on the composition or size of the HBs proteins in the SVPs. Neither deletions in preS1 nor in preS2 led to the formation of LHBs or MHBs with a lower molecular weight. Moreover, mutations abolishing the preS2 start codon had no impact on MHBs expression. We excluded variability in the viral quasispecies as an explanation for the unexpected MHBs and LHBs pattern from deep sequencing analyses, as these mutations were mainly the major variants of the quasispecies. However, in vitro analyses of a GTA genome harboring a large preS1 deletion proved that this deletion results in a truncated form of LHBs as reflected by a decreased molecular weight when expressed in vitro in comparison to our observation in the patient infected with this variant. In addition, a strong specific LHBs signal was detected in a serum sample from this patient using a preS1-specific antibody, although the preS1 deletion abolishes the binding site of this antibody as observed in vitro. Therefore, in the analyzed patients harboring preS1/preS2 mutations, LHBs and MHBs must be expressed from a genetic source that is different from the secreted viral genomes in which these mutations were detected. The nuclear cccDNA has been reported to serve as a template for the synthesis of the pregenomic RNA, which is an intermediate for viral replication and virion assembly [2]. Therefore, secreted HBV DNA is derived from cccDNA. Also, in HBe-negative patients serum HBV DNA was observed to reflect quantitative amounts of cccDNA [26]. Thus, while the viral genomes analyzed in our study were derived from cccDNA, MHBs and LHBs must be derived from a different source, which is most likely the integrated DNA. This is in line with 2 studies that showed no correlation between cccDNA amounts and HBsAg serum levels in HBeAg-negative patients [26, 27], while in HBeAg-positive patients a strong correlation was found [27]. Both, our data and these studies suggest that HBV envelope proteins can be expressed in adequate amounts also from the integrated DNA in HBeAg-negative chronic HBV-infected patients. If HBsAg from integrated HBV genomes also transcomplements viral particle synthesis in addition to the formation of SVPs is uncertain. One potential hypothesis could be that transcomplementation occurs by these intact surface proteins from integrated HBV genomes enveloping nucleocapsids that harbor the defective HBV genome derived from cccDNA. However, as absence of MHBs and certain LHBs deletions can be tolerated for virion production, virions with mutated MHBs and LHBs may exist together with wild-type SVPs expressed by other cells. But if replication-active cccDNA coexists with HBsAg expressing integrated HBV DNA within one cell, a transcomplementation of intact HBsAg in the envelope of the virions could occur. HBsAg expression from integrated DNA was also assumed in a recent trial exploring an siRNA targeting the cccDNA in HBV-infected patients. In this trial, HBsAg was not sufficiently reduced by treatment with the siRNA in HBeAg-negative patients, while a sufficient reduction was observed in HBeAg-positive chronically HBV-infected patients [28]. The authors concluded that HBsAg was derived from integrated DNA instead of cccDNA and was therefore not sufficiently reduced by the cccDNA-specific siRNA. However, loss of HBsAg, which is also regarded as a functional cure, is generally considered an important treatment goal in patients with chronic HBV infection and several novel treatment concepts also targeting HBsAg secretion are in preclinical and clinical evaluation [13]. Thus, integrated DNA, as a potential and most likely highly productive HBsAg source, has to be considered for new antiviral strategies in HBeAg-negative patients in order to induce HBsAg loss in a higher number of patients. In conclusion, HBsAg composition and morphology of subviral particles differ among HBV genotypes A–E. PreS mutations and deletions do affect HBsAg expression in vitro but no detectable effect of the observed genetic changes on the composition or size of the HBs proteins were found in our patients. Our data strongly suggest that HBsAg can be expressed in adequate amounts also from the integrated DNA in HBeAg-negative chronic HBV-infected patients. This must be considered when developing novel treatment strategies in these patients. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This work was supported by Deutsches Zentrum für Infektionsforschung (Thematic Translational Units Hepatitis) (C. S.); Deutsche Forschungsgemeinschaft (grant numbers PE 2403/2-1 (K. P.) and HI 858/12-1 (E. H.); and a grant from the Ellen Schairer-Stiftung (K. P.). Potential conflicts of interest. J. V. reports consultancies and speaker: Abbott, AbbVie, Gilead, BMS, Medtronic, Merck/MSD, and Roche. C. S. reports consultancies and speaker: Abbvie, Gilead, BMS, Intercept, Janssen, and Merck/MSD; and research support: Gilead. S. Z. reports consultancies: Abbvie, BMS, Gilead, Intercept, Janssen, and Merck/MSD. All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. World Health Organization . Hepatitis B fact sheet, updated April 2017 . http://www.who.int/mediacentre/factsheets/fs204/en/. Accessed 16 March 2018 . 2. Schädler S , Hildt E . HBV life cycle: entry and morphogenesis . Viruses 2009 ; 1 : 185 – 209 . Google Scholar CrossRef Search ADS PubMed 3. Heermann KH , Goldmann U , Schwartz W , Seyffarth T , Baumgarten H , Gerlich WH . Large surface proteins of hepatitis B virus containing the pre-s sequence . J Virol 1984 ; 52 : 396 – 402 . Google Scholar PubMed 4. Patient R , Hourioux C , Roingeard P . Morphogenesis of hepatitis B virus and its subviral envelope particles . Cell Microbiol 2009 ; 11 : 1561 – 70 . Google Scholar CrossRef Search ADS PubMed 5. Jiang B , Himmelsbach K , Ren H , Boller K , Hildt E . Subviral hepatitis B virus filaments, like infectious viral particles, are released via multivesicular bodies . J Virol 2015 ; 90 : 3330 – 41 . Google Scholar CrossRef Search ADS PubMed 6. Cornberg M , Wong VW , Locarnini S , Brunetto M , Janssen HLA , Chan HL . The role of quantitative hepatitis B surface antigen revisited . J Hepatol 2017 ; 66 : 398 – 411 . Google Scholar CrossRef Search ADS PubMed 7. Cornberg M , Protzer U , Petersen J et al. ; AWMF . Prophylaxis, diagnosis and therapy of hepatitis B virus infection - the German guideline . Z Gastroenterol 2011 ; 49 : 871 – 930 . Google Scholar CrossRef Search ADS PubMed 8. European Association for the Study of the Liver . EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection . J Hepatol 2017 ; 67 : 370 – 98 . CrossRef Search ADS PubMed 9. Terrault NA , Bzowej NH , Chang KM , Hwang JP , Jonas MM , Murad MH ; American Association for the Study of Liver Diseases . AASLD guidelines for treatment of chronic hepatitis B . Hepatology 2016 ; 63 : 261 – 83 . Google Scholar CrossRef Search ADS PubMed 10. Sarin SK , Kumar M , Lau GK et al. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update . Hepatol Int 2016 ; 10 : 1 – 98 . Google Scholar CrossRef Search ADS PubMed 11. Lucifora J , Protzer U . Attacking hepatitis B virus cccDNA–the holy grail to hepatitis B cure . J Hepatol 2016 ; 64 : 41 – 8 . Google Scholar CrossRef Search ADS 12. Tu T , Budzinska MA , Shackel NA , Urban S . HBV DNA integration: molecular mechanisms and clinical implications . Viruses 2017 ; 9 : E75 . Google Scholar CrossRef Search ADS PubMed 13. Durantel D , Zoulim F . New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus . J Hepatol 2016 ; 64 : 117 – 31 . Google Scholar CrossRef Search ADS 14. Kuhnhenn L , Jiang B , Kubesch A , et al. Impact of HBV genotype and mutations on HBV DNA and qHBsAg levels in patients with HBeAg-negative chronic HBV infection. Aliment Pharmacol Ther. 2018;1–13 . 15. Riveiro-Barciela M , Bes M , Rodríguez-Frías F et al. Serum hepatitis B core-related antigen is more accurate than hepatitis B surface antigen to identify inactive carriers, regardless of hepatitis B virus genotype . Clin Microbiol Infect 2017 ; 23 : 860 – 7 . Google Scholar CrossRef Search ADS PubMed 16. Hassemer M , Finkernagel M , Peiffer KH et al. Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes . Virology 2017 ; 502 : 1 – 12 . Google Scholar CrossRef Search ADS PubMed 17. Ausubel F , Brent R , Kingston R , Moore D , Seidman J , Smith J. Current protocols in molecular biology . In: Gallagher SR (unit 10.2); Sasse J, Gallagher SR (unit 10.7); Gallagher S, Winston SE, Fuller SA, Hurrell JGR (unit 10.8), eds. John Wiley , 2003 ; Supplement 66. 18. Kucinskaite-Kodze I , Pleckaityte M , Bremer CM et al. New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen . Virus Res 2016 ; 211 : 209 – 21 . Google Scholar CrossRef Search ADS PubMed 19. Deepen R , Heermann KH , Uy A , Thomssen R , Gerlich WH . Assay of preS epitopes and preS1 antibody in hepatitis B virus carriers and immune persons . Med Microbiol Immunol 1990 ; 179 : 49 – 60 . Google Scholar CrossRef Search ADS PubMed 20. Dietz J , Schelhorn SE , Fitting D et al. Deep sequencing reveals mutagenic effects of ribavirin during monotherapy of hepatitis C virus genotype 1-infected patients . J Virol 2013 ; 87 : 6172 – 81 . Google Scholar CrossRef Search ADS PubMed 21. Susser S , Flinders M , Reesink HW et al. Evolution of hepatitis C virus quasispecies during repeated treatment with the NS3/4A protease inhibitor telaprevir . Antimicrob Agents Chemother 2015 ; 59 : 2746 – 55 . Google Scholar CrossRef Search ADS PubMed 22. Sominskaya I , Pushko P , Dreilina D , Kozlovskaya T , Pumpen P . Determination of the minimal length of preS1 epitope recognized by a monoclonal antibody which inhibits attachment of hepatitis B virus to hepatocytes . Med Microbiol Immunol 1992 ; 181 : 215 – 26 . Google Scholar CrossRef Search ADS PubMed 23. Pfefferkorn M , Bohm S , Schott T et al. Quantification of large and middle proteins of hepatitis B virus surface antigen (HBsAg) as a novel tool for the identification of inactive HBV carriers . Gut [published online ahead of print 26 September, 2017]. doi: 10.1136/gutjnl-2017-313811 . 24. Sozzi V , Walsh R , Littlejohn M et al. In vitro studies show that sequence variability contributes to marked variation in hepatitis B virus replication, protein expression, and function observed across genotypes . J Virol 2016 ; 90 : 10054 – 64 . Google Scholar CrossRef Search ADS PubMed 25. Rydell GE , Prakash K , Norder H , Lindh M . Hepatitis B surface antigen on subviral particles reduces the neutralizing effect of anti-HBs antibodies on hepatitis B viral particles in vitro . Virology 2017 ; 509 : 67 – 70 . Google Scholar CrossRef Search ADS PubMed 26. Lin LY , Wong VW , Zhou HJ et al. Relationship between serum hepatitis B virus DNA and surface antigen with covalently closed circular DNA in HBeAg-negative patients . J Med Virol 2010 ; 82 : 1494 – 500 . Google Scholar CrossRef Search ADS PubMed 27. Thompson AJ , Nguyen T , Iser D et al. Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers . Hepatology 2010 ; 51 : 1933 – 44 . Google Scholar CrossRef Search ADS PubMed 28. Wooddell CI , Yuen MF , Chan HL et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg . Sci Transl Med 2017 ; 9 : eaan0241 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of Infectious DiseasesOxford University Press

Published: Mar 8, 2018

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