A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B

Abstract

Background & aims: Circulating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of covalently closed circular DNA (cccDNA) and may help to evaluate HBV treatment activity. Different research assays have been proposed and, although two PCR-based research use only investigational assays have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays.

Methods: HBV RNA-producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 unit length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP domain (Y63F) of the polymerase, and the ε-loop of the pregenomic (pg)RNA (mutation A1G).

Results: The clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virion-like particles and only a minority in small extracellular vescicles. Nanopore sequencing of 5'RACE products shows that the majority of the Huh7-3D29-secreted HBV RNAs start at the 5' end of pgRNA and pgRNA-derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from one flask.

Conclusion: We generated a clonal cell line that produces high quantities of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assay calibration.

Impact and implications: Several investigational assays and two research use only assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of covalently closed circular DNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients' sera (e.g. pgRNA, HBx transcripts). This new RNA standard can be used to calibrate all PCR-based assays for circulating HBV RNA quantification to evaluate, in a non-invasive manner, the size of the transcriptionally active cccDNA pool and the activity of novel strategies aimed at curing HBV infection.

Keywords: biomarker; chronic hepatitis B (CHB); hepatitis B virus (HBV); pregenomic RNA (pgRNA); standard.

Graphical abstract

Fig. 1

Strategies to generate an HBV RNA standard. (A) Viral mutations selected for the generation of cell lines and their expected effect on viral parameters. ¹, , , ; ²; ³, , ⁴ ↘: decrease, (B) pTriEX plasmid based plasmids containing one of the five 1.1-unit length HBV genotype D genomes (YMAA (1); Y63F (2); YMAA + Y63F (3); A1G + YMAA (4); A1G + Y63F (5) or the 1.1 WT HBV genome. (C) Two-step ddPCR quantification of HBV DNA and 3.5 kb HBV RNA species in the supernatants of the HBV-Huh7 polyclonal cell lines. Error bars represent standard deviation of 3 technical replicates from each of 3 distinct cell passages, which served as biological replicates. ddPCR, digital droplet PCR; NA, nucleic acid; WT, wild-type.

Fig. 2

HBV nucleic acid secretion profiles of selected HBV-Huh7 stable clones. Two-step ddPCR quantification of secreted HBV DNA and 3.5 kb HBV RNA species from Huh7 clonal cell lines at different passages and freezing/thawing cycles. WT = WT genotype D HBV. 3D = genotype D HBV genome carrying the ε-loop A1G mutation and the HBV polymerase catalytic site YMAA mutation. 5D = genotype D HBV genome carrying the ε-loop A1G mutation and the Y63F mutation in the TP domain of the HBV polymerase. The values following the WT, 3D and 5D label indicate the clone number. The p followed by a value correspond to the number of passages in cell culture. Error bars represent standard deviation of two technical replicates from each of three distinct cell passages, which served as biological replicates. ddPCR, digital droplet PCR; WT, wild-type.

Fig. 3

Sucrose/iodixanol gradient characterization of HBV nucleic acids secreted in HBV-Huh7 3D29 clone culture supernatant. Upper panels: Distribution of hepatitis B viral particle-associated antigens and DNA/RNA in sucrose/iodixanol gradient for WT18 (A) and 3D29 (B) clonal cell lines. Total HBV DNA and total HBV RNA were quantified in the sucrose/iodixanol gradient fraction by two-step ddPCR, as detailed in the Methods section. Lower panels: Western Blot detection of viral HBc protein and CD9 sEV marker. ddPCR, digital droplet PCR; sEV, small extracellular vesicle; WT, wild-type.

Fig. 4

Characterization of HBV RNAs in Huh7-3D29 cell culture supernatant. (A) PAGE analysis of the 5’RACE performed on cell extracts and culture supernatants of the Huh7-3D29 clone. Canonical HBV RNA positions are indicated on the left. (B) Nanopore sequencing of the 5’RACE products in the cell culture supernatants (upper panel) and cellular extract of Huh7-3D29 cells (lower panel). 85,497 and 70,588 reads were generated for the Huh7-3D29 cellular extract and cell culture supernatants, respectively. Left panels are the number of reads according to their starting position after alignment on the HBV genome. Right panels indicate the proportion (%) of reads associated with each HBV canonical promoter. MW, molecular weight; 5’RACE, 5’ rapid amplification of cDNA ends.

Fig. 5

Validation of Huh7-3D29 cell culture supernatant as a standard for HBV RNA quantification assays. (A) The comparison of 10-fold dilutions of Huh7-3D29 supernatant and armored RNA performance did not show significant differences (paired t test, p = 0.54). Amplification curves of the arRNA (left panel) and 3D29 HBV RNA (right panel) in the Roche Manual workflow assay. Middle panel: Ct values and corresponding concentration in copies/ml. Validation of samples parametric distribution by Shapiro-Wilk test. (B) Performance of serial dilutions of cell supernatants from three Huh7-3D29 passages and of the HBV arRNA in an in house ddPCR. (C) Huh7-3D29 HBV RNA quantification with and without DNAse treatment (Wilcoxon test, p = 0.25). Validation of samples non-parametric distribution by Shapiro-Wilk test. Error bars represent standard deviation of two technical replicates from each of three distinct cell passages, which served as biological replicates. arRNA, armored RNA; ddPCR, digital droplet PCR.