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

Alexia Paturel, Francesca Casuscelli Di Tocco, Delphine Bousquet, Marie-Laure Plissonnier, Xavier Grand, Hyosun Tak, Françoise Berby, Caroline Scholtès, Barbara Testoni, Fabien Zoulim, et al.

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## To cite this version:

Alexia Paturel, Francesca Casuscelli Di Tocco, Delphine Bousquet, Marie-Laure Plissonnier, Xavier Grand, et al. A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B. JHEP Reports Innovation in Hepatology, 2024, 6 (10), pp.101124. 10.1016/j.jhepr.2024.101124. hal-05021460

HAL Id: hal-05021460

https://hal.science/hal-05021460v1

Submitted on 4 Apr 2025

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

## Authors

Alexia Paturel, Francesca Casuscelli di Tocco, Delphine Bousquet, ..., Barbara Testoni, Fabien Zoulim, Massimo Levrero

## Correspondence

massimo.levrero@inserm.fr (M. Levrero).

## Graphical abstract

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## Highlights:

* Circulating HBV RNA reflects the transcriptional activity of cccDNA and may help to evaluate HBV treatment activity.

* Lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation.

* The clonal Huh7-3D29 cell line produces an RNA-based standard for calibration of PCR-based circulating HBV RNA assays.

* Huh7-3D29 cells show a high, stable, and up-scalable secreted RNA yield.

* The 3D29 RNA standard contains the major HBV RNA species found in HBV patient sera (e.g. pgRNA, HBx transcripts).

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

Alexia Paturel<sup>1,2,3</sup>, Francesca Casuscelli di Tocco<sup>1,2,3</sup>, Delphine Bousquet<sup>1,2,3</sup>, Marie-Laure Plissonnier<sup>1,2</sup>, Xavier Grand<sup>1,2,3</sup>, Hyosun Tak<sup>1,2,3</sup>, Françoise Berby<sup>1,4</sup>, Caroline Scholtès<sup>1,2,3,5</sup>, Barbara Testoni<sup>1,2</sup>, Fabien Zoulim<sup>1,2,3,4</sup>, Massimo Levrero<sup>1,2,3,4,6,*</sup>

JHEP Reports 2024. vol. 6 | 1–11

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Check for updates

**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.

© 2024 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

HBV remains a major public health problem worldwide, despite the availability of an effective vaccine and antiviral therapies. 296 million people were living with chronic hepatitis B (CHB) infection in 2019, with 0.5-1 million dying annually because of HBV-related advanced liver disease.¹ Long-term treatment with anti-HBV nucleos(t)side analogues (NUCs) suppresses viral replication, liver inflammation and disease progression.² However, only a small proportion of patients achieve a *functional HBV cure*, defined by a sustained off-treatment loss of hepatitis B surface antigen (HBsAg) that is associated with an improved long-term clinical outcome and the ability to stop treatment with very low rates of disease reactivation.³,⁴ Innate and adaptive antiviral immune responses control the residual covalently closed circular DNA (cccDNA) reservoir.⁵ A *complete HBV cure* with eradication of all cccDNA in the liver and no residual risk of viral reactivation, is rarely, if ever, achieved with NUCs. The development of new antiviral therapies targeting the cccDNA reservoir is a major move towards *HBV cure*.⁵ Several new molecular entities (NMEs) with direct antiviral or

immunomodulatory activities are being developed, with the aim of inducing cure with a finite treatment duration.

Direct assessment of cccDNA levels and activity requires liver biopsy and cccDNA quantification remains technically challenging.⁶⁻⁸ To assist the clinical development of these NMEs, there is an urgent need for the development of non-invasive biomarkers that adequately reflect the size and transcriptional activity of intrahepatic cccDNA. HBV DNA and HBsAg, the currently used biomarkers to define HBV endpoints have several limitations in this setting: i) serum HBV DNA is the best indicator of the antiviral activity of a new treatment in naive patients but it is already suppressed in NUC-treated patients; ii) HBsAg loss can at the same time act as a marker of target engagement, reflecting the direct activity of specific NMEs and the endpoint of all new therapies; iii) the predictive value of different thresholds of HBsAg decrease remains to be validated and the kinetics of HBsAg decrease are often too slow to allow for early outcome prediction in clinical trials. In addition, HBsAg may be expressed from the viral genome integrated into the host genome and thus its level may not fully reflect the

<footnote>* Corresponding author. Address: Institute of Hepatology Lyon (IHL), Cancer Research Center of Lyon (CRCL) - INSERM U1052, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France.</footnote>

<footnote>E-mail address: massimo.levrero@inserm.fr (M. Levrero).</footnote>

<footnote>https://doi.org/10.1016/j.jhepr.2024.101124</footnote>
transcriptional activity of cccDNA.<sup>9,10</sup> Thus, new biomarkers
would be useful: a) to better characterize patients in the
different phases of HBV infection and disease; b) to predict
response to interferon (IFN) at an early stage, so that treatment
is only extended in patients with a high probability of achieving
a functional cure; c) to identify patients that could discontinue
NUCs without risk of relapse; d) to assess target engagement
and antiviral activity in patients receiving NMEs; e) to predict
HBsAg response, i.e. functional cure, in patients receiving
NMEs.<sup>7,11</sup>

Several studies have identified circulating HBV RNAs as a
biomarker reflecting the transcriptional activity of cccDNA<sup>12,13</sup>
and have shown that circulating HBV RNA may help monitor
CHB infection.<sup>11,14,15</sup> Although the potential relevance of
circulating HBV RNAs as a biomarker is well established, a
major limitation is the lack of standardized protocols (e.g.,
sample extraction, reverse transcription, choice of primers for
amplification, genotype inclusivity, specificity for RNA). Inves-
tigational research use only (RUO) assays to quantify circu-
lating HBV RNAs have been developed by industry.<sup>16-18</sup>
Different standards have been used for the two RUOs: a) the
World Health Organization (WHO) International Standard (IS) for
HBV DNA,<sup>16</sup> originally established to be used as a calibrator for
HBV DNA nucleic acid amplification technique-based assays;
<sup>19-24</sup> b) a synthetic armored RNA (arRNA) derived from the 3'
end of HBV pregenomic RNA (pgRNA).<sup>16-18</sup> The appropriate-
ness of the WHO HBV DNA standard has been challenged.<sup>25</sup>
Indeed, it is questionable whether an HBV RNA unit is equiv-
alent to an international HBV DNA unit defined by the plasma
derived WHO HBV DNA standard that also contains circulating
HBV RNA.<sup>25</sup> Conversely, arRNA synthesis is unpractical and
costly for routine use. The lack of a unique, recognized cali-
brator for circulating HBV RNA quantification remains a
potentially significant limitation to compare results generated
by different assays.

Herein, we describe the generation of a stable clonal cell line
producing an RNA-based standard for the calibration of
circulating HBV RNA assays that are used to evaluate the size
of the transcriptionally active cccDNA pool in a non-invasive
manner and the activity of novel strategies aimed at curing
HBV infection.

Materials and methods

Cell lines

The human Huh7 cell line was cultured at 37 °C in a humidified
atmosphere containing 5% CO₂ in Dulbecco's modified Eagle
medium supplemented with 10% Hyclone fetal clone II serum
1% GlutaMax (Gibco), 1% Penicillin/Streptomycin (Gibco), 1%
sodium pyruvate (Gibco), 1% non-essential amino acids
(NEAA, Gibco).

Plasmids

The HBV RNA-producing cell lines was generated by trans-
fecting a pTriEX plasmid derivative containing 1.1-unit length
WT and mutated HBV DNA genomes in Huh7 cells. In total,
three mutations were selected: YMAA and Y63F in the poly-
merase open reading frame, and A1G in the ε-loop region. Five
HBV mutated genomes and a wild-type (WT) HBV genome of
genotype D have been generated and inserted into the pTriEX-

Bsd vector: a plasmid containing the WT 1.1 unit HBV genome, two plasmids containing the 1.1 unit HBV genome carrying the YMAA mutation or the Y63F mutation and three plasmids containing the 1.1 unit HBV genome carrying the following combinations of mutations: YMAA + Y63F, A1G + YMAA, and A1G + Y63F. All plasmids include the blasticidin resistance gene for cell culture selection. WT and mutated 1.1-unit HBV genomes were synthetized and the plasmids produced and controlled by sequencing by GenScript®.

Cell transfection and culture

Huh7 cells were seeded at 60-80% confluency and were allowed to adhere overnight. Cells were then transfected with the indicated amounts of total plasmid DNA with the TransIT®-2020 kit (Mirus) in serum-free Opti-MEM medium (Life Technologies), following the manufacturer's instructions. After 12h, the transfection medium was removed and was replaced with fresh medium. Transfected cells were grown and amplified in selective growth media (Huh7 medium described above) supplemented with 4 µg/ml of Blasticidin.

Clonal cell line isolation and expansion

Colony formation was obtained after serial dilution of Huh7
polyclonal cell population from 1 × 10⁴ cells to 100 cells per
150 cm² petri dish. Cell plates were incubated at 37 °C with 5% CO₂ for 20 days to allow colony formation and growth. Using single channel pipettor, single colonies were transferred to 1 ml PBS. Cells were pelleted by centrifugation for 5 min at 900 rpm and resuspended in 50 µl of 0.25% trypsin-EDTA (Gibco) and kept for 5 min at 37 °C. Cells were then plated in 24 wells plates with 1 ml fresh Huh7 media. Clonal cell lines were amplified under blasticidin selection. Clones to be further carried on were chosen based on cell growth, HBV RNA/HBV DNA ratio, and HBV RNA productivity.

High scale supernatant production

To test the stability over time of the HBV RNA secretory
phenotype and to perform higher scale supernatant collection
to produce the HBV RNA standard, cells were seeded (25 × 10⁶
cells/175 cm² flask) and cultured overnight before change of
media containing 2.5% DMSO. To collect large quantities of
HBV RNAs, cells were kept under standard culture conditions
for 9 days. Every 3 days, supernatant was collected, and fresh
media was added. After 9 days, all collected supernatants were
pooled and centrifuged for 5 min at 1,500 rpm to remove any
cellular debris.

Ultracentrifugation, DNA and RNA isolation

Pooled cell culture supernatants were ultracentrifuged for 5 h at
25,000 rpm over a 4 ml 20% w/v sucrose cushion. After ul-
tracentrifugation, the supernatant was removed, and the virus-
containing pellet was resuspended in 600 µl of PBS 1X. 28.5 µl
aliquots (28.5 µl out of 600 µl) of each resuspended pellet was
completed to 200 µl with PBS 1X. DNA and RNA from resus-
pended pellets were extracted using High Pure Viral Nucleic
Acid (Roche, Diagnostics) according to the manufacturers' in-
structions. The same final volume of extracted nucleic acid
elution (40 µl) was used for DNA or RNA analysis. RNA samples
were treated with RQ1 RNase-Free DNase (Promega, Cat# M6101) for 30 min at 37 °C and stored until use.

## HBV nucleic acids quantification by 2-step droplet digital PCR (ddPCR)

For total HBV DNA analysis 4 µl of eluted DNA were used. For HBV RNA analysis, 4 µl of DNase-treated RNA was reverse transcribed and amplified using the SuperScript™ IV VILO™ Master Mix (Invitrogen, Cat #11766500). The 22 µl amplification mixtures comprise 11 µl of 2X ddPCR Supermix™ for probes (no dUTP) (Bio-Rad), 1.1 µl of primers and probe mix, and 5 µl of either DNA or cDNA. Nucleic acid inputs were adjusted to have acceptable rates of *negative events*: samples were diluted 1:5. Probes and primers include: the HBV Pa03453406_s1 primers and probe set (Thermofischer) for both total HBV DNA and total HBV RNA quantification and the forward primer ggagtggtaggtcgactcct, reverse primer agattgagatcttctgcgac and probe aggcgaggtcccctagaagaagactcc for 3.5 kb HBV RNA species quantification. Droplet formation was carried out using a QX200 AutoDG droplet generator. Subsequent amplifications were performed in the C1000 Touch™ deep-well thermal cycler (Bio-Rad) with a ramp rate of 2 °C/s and the lid heated to 105 °C, according to the Bio-Rad recommendations. First, the enzyme was activated at 95 °C for 10 min followed by 40 cycles of denaturation at 94 °C for 30 s and 60 °C for 1 min. The enzyme was deactivated at 98 °C for 10 min and the reaction was kept at 4 °C.

## Quantification of HBV RNA with the Roche MWF investigational assay

The Roche Manual Workflow (MWF) is a manual version developed for research purposes of the cobas® 6800/8800 Automated Investigational Assay (IA) for the detection and quantification of circulating HBV RNAs in chronic HBV patients.<sup>17,18</sup> MWF uses identical chemistry and PCR primers/probe sets as the cobas® 6800/8800 automated IA but does not require high throughput and automation. Results obtained with this manual assay were highly correlated with those obtained with cobas HBV RNA. HBV RNA assay characteristics are the following: i) linear results between 10 and 10<sup>7</sup> copies/ml in clinical samples of several HBV genotypes; ii) precision and reproducibility with standard deviation below 0.15 log<sub>10</sub> copies/ml and coefficients of variation below 5% throughout the linear range; minimal impact (<0.3 log<sub>10</sub> copies/ml) of HBV DNA on HBV RNA quantification at DNA:RNA ratios of up to approximately 10<sup>6</sup>. HBV RNA quantification was calibrated using a synthetic arRNA containing 435 bp derived from the 3' end of HBV pgRNA, packaged in MS2-phage (kindly provided by Roche Diagnostics, Pleasanton, CA). arRNA was quantified by ddPCR (Bio-Rad) using a primers/probe set in the precore/core region. HBV RNA quantification with the MWF was performed according to the manufacturer's protocol.

## Iodixanol/sucrose density gradient centrifugation

300 ml of supernatants were collected and centrifuged at 1,500 x g for 15 min at room temperature. Cellular debris were removed by filtration through a 0.22 µm filter (Merck Millipore, KGaA, Darmstadt, Germany). Viral particles were concentrated using Amicon® Pro Purification System with 100 kDa filter.

Concentrated supernatants were then ultracentrifuged at 110,000 x g for 2h at 4 °C. The pellets were washed with 8 ml of PBS and a second ultracentrifugation was performed at 110,000 x g for 2h at 4 °C. Pellets were resuspended in 2.5 ml of 10% iodixanol solution. 10%, 20%, 30% and 40% iodixanol solutions were prepared by mixing Optiprep™ (Axis Shield) with buffer containing 0.25 M sucrose, 10 mM Tris at pH 8.0, and 1 mM EDTA, with final pH 7.4. Resuspended EVs pellets were layered on the top of the gradient and then subjected to ultracentrifugation in a SW41-Ti Rotor tube (Beckman) for 6 h at 4 °C at 110,000 x g. Twelve fractions of 1 ml were recovered and analyzed separately.

## Viral protein detection

ELISA tests for HBeAg and HBsAg detection in cell supernatants were performed according to the manufacturer's protocol using the CLIA kits from Autobio Diagnostic.

Viral proteins were detected in the density gradient fractions by Western blot. Fractions were mixed with Laemmli buffer and heated at 95 °C for 5 min. Proteins were migrated in 4-20% mini-PROTEAN® TGX stain-FreeTM Precast Gel (Bio-Rad Laboratories) and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked 1 h with 5% milk or BSA (Sigma) in TBS (1 x Tris Buffer Saline (Sigma)) and stained with primary antibodies in blocking buffer overnight at 4 °C and stained with HRP-conjugated secondary antibodies (1/50,000) for 1 h at room temperature. Detection was performed using Clarity or Clarity Max Western ECL and the ChemiDoc XRS system (Bio-Rad).

## 5'RACE analysis

5' rapid amplification of cDNA ends (5'RACE) was performed as previously described in.<sup>26</sup> Briefly, RNAs were isolated using a guanidinium thiocyanate-phenol-chloroform extraction protocol (TRI reagent (Sigma)). 5'RACE was essentially performed as described in the GeneRacer Kit manual (ThermoFisher Scientific) except Tobacco Acid Pyrophosphatase was substituted by RNA 5' Pyrophosphohydrolase (New England Biolabs) and SuperScript reverse transcriptase III by SuperScript reverse transcriptase IV (ThermoFisher Scientific). The reverse transcription reaction was performed using 3' HBV specific Gsp1 primer 5'-TTAGGCAGAGGTGAAAAAAGTTG-3'. For the 5'RACE PCR reaction Prime Star super mix DNA Polymerase (TAKARA bio), GeneRacer 5' primer and HBV specific nested primer Gsp1 5'-TTAGGCAGAGGTGAAAAAAGTTG-3' were used. 5'RACE PCR was run in a C100 Touch thermocycler (Bio-Rad) using the following PCR program: initial denaturation step 98 °C 3 min >5x (98 °C 10 s; 72 °C 3 min) >5x (98 °C 10 s; 70 °C 3 min) >25x (98 °C 10 s; 64,4 20 s; 72 °C 3min) >72 °C 10min.

## MinION sequencing, data processing and bioinformatics analysis

5'RACE Samples were further prepared individually following the recommendations of the Nanopore protocol for the SQK-PBK004. Base calling was performed with Guppy (v. 6.4.6)<sup>27</sup> and simultaneously filtered for base called reads with PHRED quality >7 and trimmed for technical adapters. Further quality controls of reads before and after mapping were performed with pycoQC (v. 2.5.2).<sup>28</sup> Median PHRED score is 11.455 for passed reads and
99.6 % of reads are conserved with acceptable quality. Raw reads were trimmed for technical adapters using Porechop (https://github.com/rrwick/Porechop). Reads containing 5'RACE adapter sequence were extracted with seqkit (v. 2.1.0)²⁹ using the grep function, with 10% mismatch allowed due to error rate of Oxford MinION Nanopore sequencing. Reads were trimmed for the 5'RACE adapter sequence using cutadapt (v. 3.5).³⁰ Trimmed reads were mapped to the genomic sequence corresponding to the full-length preCore RNA sequence (from the transcription start site [TSS] to the canonical HBV poly-A, derived from HBV genome, strain ayw, NC_003977.2) with Minimap2 (v. 2.21-r1071)³¹,³² using the spliced long reads option (-ax splice). Sam files were filtered, binarized and sorted with Samtools.³³ Start positions were extracted from genomic alignment using Samtools, splice junctions were identified using Nanosplicer (v. 1.0),³⁴ then quantification and graphical representations were produced using R scripts (v. 4.1.2).

## Statistics

Statistical tests were performed using GraphPad Prism v7.05 software. Pairwise comparisons were analyzed using the Wilcoxon signed-rank test for non-parametric data and paired t test for parametric data. The comparison of more than three groups was performed using the Kruskal-Wallis test. Shapiro-Wilk normality test was performed to assess the normality of the parameter's distribution (GraphPad prism). Differences were considered significant at confidence levels greater than 95% ($p \le 0.05$).

## Results

### Generation of cell lines carrying modified HBV genomes and inverted DNA/RNA secretion

To generate a continuous source of HBV RNA to be used to calibrate HBV RNA quantification assays, we established a stable clonal cell line carrying a modified HBV genome and producing and secreting RNA-containing viral particles similar to those found in patient sera. To this aim we selected three candidate mutations in the catalytic and TP domains of viral polymerase, and in the $\varepsilon$-loop of the pgRNA that are expected, alone or in combination, to impact on pgRNA encapsidation, reverse transcription, relaxed circular DNA (rcDNA) formation and global viral production (Fig. 1A).

The YMDD motif is in the catalytic site of the HBV Pol and mutations in this motif are involved in resistance to anti-HBV nucleoside analogues.³⁵⁻³⁸ Different mutations in the YMDD domain also result in a 20% to 90% decrease in viral DNA synthesis.³⁶,³⁸ The specific mutations selected for this study change the YMDD motif in YMAA (D540A/D541A).

The Polymerase tyrosine 63 residue (Y63) in the TP domain is required for the priming of reverse transcription by providing the hydroxyl group necessary for the covalent binding between the first nucleotide of the negative strand of DNA and the viral polymerase.³⁹⁻⁴¹ Mutation of tyrosine 63 to a phenylalanine (Y63F) has been shown to result in a strong increase in HBV RNA virions containing pgRNA.⁴² The A1G or Q18R mutation in the pgRNA stem loop structure of the encapsidation signal has been shown to induce a drastic

decrease in viral DNA synthesis by interfering with the priming of reverse transcription.⁴³

A WT and five mutant 1.1 unit length HBV genotype D genomes (YMAA (1); Y63F (2); YMAA + Y63F (3); A1G + YMAA (4); A1G + Y63F (5)) were synthesized and cloned into a pTriEX vector plasmid (Fig. 1B). After transfection and blasticidin selection in Huh7 cells, the six polyclonal cell lines were analyzed for HBV DNA and HBV RNA in cell extracts and cell supernatants. The YMAA + Y63F (3) and A1G + Y63F (5) polyclonal cell lines displaying the desired secretory phenotype with an inversion of the HBV DNA/RNA ratio in cell supernatants, lower DNA secretion and lower intra-experiment variability (e.g. SD values) were selected for the generation of clonal cell lines (Fig. 1C).

### Secretory phenotype of selected clonal cell lines

Next, the WT, (3) YMAA + Y63F and (5) A1G + Y63F polyclonal cell lines were plated at low density for colony formation. After 20 days of blasticidin selection, 24 clones for each polyclonal cell line were isolated, expanded and phenotyped for HBV DNA/RNA secretion. Clones Huh7-WT18 (carrying the integrated WT HBV genome of genotype D), Huh7-5D21 (carrying a mutation in both the $\varepsilon$-loop of the pgRNA (A1G) and the TP domain of the polymerase (Y63F), see Fig. 1A), Huh7-3D29 and Huh7-3D35 (carrying the mutation in the TP domain of the polymerase (Y63F) and the catalytic site of the HBV polymerase (YMAA), see Fig. 1A), were selected based on cell growth, HBV RNA/DNA ratio and HBV RNA production (data not shown) and carried on for further characterization and expansion. At 9 days post plating, Huh7-WT18 cells secreted 15-fold more HBV DNA than 3.5 kb HBV RNA (1.08 × 10⁷ copies of HBV DNA/ml vs. 0.7 × 10⁶ copies of 3.5 kb HBV RNA/ml). Huh7-5D21, Huh7-3D29 and Huh7-3D35 clonal cell lines showed high HBV RNA levels compared to HBV DNA levels in cell supernatant, with an inversion of the secreted HBV DNA/RNA ratio (Fig. 2, left panel). 3.5 kb HBV RNA secretion levels were 1.0 × 10⁷ copies/ml in Huh7-3D35 cells, 1.6 × 10⁷ copies/ml in Huh7-5D21 cells and 6.02 × 10⁷ copies/ml in Huh7-3D29 cells. All three mutant clonal cell lines secreted more 3.5 kb HBV RNA than the Huh7-WT18 WT clone: 90x higher in Huh7-3D29, 27x higher in Huh7-5D21 cells and 16x higher in Huh7-3D35 cells. Notably, lamivudine treatment for 9 days resulted in a sharp reduction of secreted HBV DNA in Huh7-WT18 WT cells, a further reduction, but not a complete abrogation, of secreted HBV DNA in Huh7-3D29 cells (Fig. S1, black columns) and a slight increase in the levels of HBV DNA secreted in both the HBV RNA WT the 3D29 cells (Fig. S1, grey columns).

After a first freeze/thaw cycle (3 passages), the Huh7-5D21 and Huh7-3D29 mutant cell lines kept their RNA secretory phenotype (Fig. 2, middle panel). A decrease in secretion of all HBV nucleic acids was observed over the passages without a significant change in the HBV RNA/DNA ratio. Huh7-3D29 cells showed a 34% reduction of 3.5 kb HBV RNA secretion but kept the higher RNA/DNA ratio. After a second freeze/thaw cycle (3-4 passages), the HBV RNA secretory phenotype was conserved (Fig. 2, right panel). The Huh7-3D29 cell line again showed a higher RNA/DNA ratio with the lower residual HBV DNA (2.44 × 10⁷ RNA copies/ml vs. 2.87 × 10⁵ DNA copies/ml). Finally, the secretory phenotype of the Huh7-3D29 clone is consistent between vials issued from the same culture passage (Fig. S2).
A

<figure><img src="image_5.png" /></figure>

<table><thead><tr><th></th><th>WT</th><th>YMAA<sup>(1)</sup></th><th>A1G<sup>(2)</sup></th><th>Y63F<sup>(3)</sup></th></tr></thead><tbody><tr><td>DNA accumulation</td><td>100%</td><td>↓<sup>(4)</sup></td><td>&lt;18%</td><td>&lt;10%</td></tr><tr><td>pgRNA encapsidation</td><td>100%</td><td>≈100%</td><td>≈100%</td><td>≈100%</td></tr><tr><td>In vitro priming</td><td>100%</td><td>↓<sup>(4)</sup></td><td>↓<sup>(4)</sup></td><td>20%</td></tr></tbody></table>

B

<figure><img src="image_6.png" /><figcaption>**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. ¹³⁵⁻³⁸, ²⁴³, ³³⁹⁻⁴¹ 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.</figcaption></figure>

## Characterization of the HBV RNAs secreted by the Huh7-3D29 clonal cell line

Although the majority of circulating HBV RNAs are represented by the pgRNA-containing viral particle,²⁶,⁴²,⁴⁴⁻⁴⁸ increasing evidence suggests the presence of additional RNA species and variable amounts of circulating HBV RNAs might be carried in small extracellular vesicles (sEVs).⁴⁹⁻⁵¹ To characterize the HBV

RNA species present in Huh7-WT18, Huh7-3D29, and Huh7-3D35 cell supernatants and their compartmentalization, we performed iodixanol/sucrose density gradient ultracentrifugation⁵² followed by HBsAg quantification by ELISA, HBV DNA and HBV RNA quantification by ddPCR, and HBc and CD9 sEV marker⁵³ detection by western blot (Fig. 3). In Huh7-WT18 cell supernatants (Fig. 3A), HBsAg was detected in fractions 4-8 of
<figure><img src="image_7.png" /><figcaption>**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.</figcaption></figure>

the density gradient, with one major peak in fraction 7 (Fig. 3A, upper panel), and HBc was detected from fractions 7 to 9 (Fig. 3A, lower panels). HBV DNA was found in almost all the fractions except in fractions 1 and 2, with a peak in fraction 8 (Fig. 3A, upper panel). HBV RNA was found in fractions 6 to 9 (Fig. 3A, upper panel). Fractions 3 and 4 contain sEVs, identified by the detection of the CD9 membrane protein⁵³ (Fig. 3A, lower panels). Notably, in Huh7-WT18 cell supernatants the sEVs contained only HBV DNA (Fig. 3A, upper panel). The detection of both HBsAg and HBc identifies viral particles, while the detection of HBc only identifies naked capsids. Fractions 7 and 8, positive for both HBsAg and HBc, were positive for both HBV DNA (virions) and HBV RNA (RNA particles) (Fig. 3, upper panel). Fraction 9, positive for HBc only, contained only low levels of HBV RNA compared to HBV DNA, indicating that naked capsids mostly carry HBV DNA in WT

cells (Fig. 3A, upper panel). In Huh7-3D29 cell supernatants, HBV RNA was found in RNA particles (fraction 7 to 8) and in naked capsids (fraction 9) while the residual HBV DNA peak was found in virions (fraction 7) (Fig. 3B, upper panel). Compared to the Huh7-WT18 cell, a larger proportion of HBV RNA was found in naked capsids. This observation is consistent with the reduction of HBV DNA synthesis in the immature capsid imposed by the HBV mutations and the resulting blockade of capsid maturation. Regarding the distribution of HBsAg, HBc and CD9 in the different fractions, no differences were noticed compared to the WT clone.

To refine the characterization of the RNA species secreted by Huh7-3D29 cells, we have performed 5'RACE coupled with single molecule long-read Nanopore sequencing. Gel electrophoresis analysis of the 5'RACE products from Huh7-3D29 cells and supernatants shows that only 3.5 kb RNA species were detected in

A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /><figcaption>**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.</figcaption></figure>
cell supernatants (Fig. 4A). Nanopore sequencing of the 5'RACE products shows that: a) the secreted HBV RNA is identical to the HBV genome used for the establishment of the different cell lines; and b) the majority of HBV RNAs detected start at the 5'end of pgRNA and pgRNA-derived spliced RNAs; c) lower amounts of several HBx RNA species are present; d) PreS/S RNAs and PreS/S-derived spliced RNA are barely detectable (Fig. 4B, upper

panel). The proportion of spliced transcripts from the 3.5 kb RNA TSS is low (0.84%), with a relative enrichment of SP1 (84.2%) as the prominent species (Fig. S3A). The profile of HBV RNAs detected in the cellular fraction was very different with all canonical HBV RNA transcripts present and a majority of PreS1/PreS2/S transcripts. Spliced RNAs represent 1.77% of all transcripts derived from the 3.5 kb RNA TSS (Fig. S3B). SP1 accounts for

A

<figure><img src="image_10.png" /></figure>

B

<figure><img src="image_11.png" /></figure>

**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.
27.8% of all the spliced HBV RNAs starting at the 3.5 kb RNA TSS and 0.7% of all 3.5 kb RNAs, with several other SP variants represented (Fig. S3B). Altogether, the coupled 5'RACE - Nanopore sequencing results support the notion that the Huh7-3D29 HBV RNA-containing supernatants could be used to standardize RUO HBV RNA investigational assays¹⁶⁻¹⁸ and in house PCR-based HBV RNA assays¹³ aimed at quantifying HBV pgRNA.

## Use of RNA-containing supernatant from Huh7-3D29 cells to standardize the manual version (MWF) of the HBV RNA Roche IA

Ten-fold dilution (from $10^5$ to $10^1$ copies/ml) of HBV arRNA (Roche Diagnostics, Pleasanton, CA)¹⁷,¹⁸ and Huh7-3D29 supernatants, processed as described in the Materials and Methods, have been quantified using the HBV RNA Roche MWF kit.¹⁷,¹⁸ The amplification curves obtained after quantitative reverse-transcription PCR from the MWF showed very similar profiles (Fig. 5A, left and right panels). Furthermore, no significant difference was found between CTs of the 10-fold diluted arRNA and Huh7-3D29 cell supernatants (Fig. 5A, middle panel). Next, we compared the performance of serial dilutions of cell supernatants from three Huh7-3D29 passages and of the HBV arRNA using an in house ddPCR (see Materials and Methods) (Figs 5B and S6). HBV RNA quantification of Huh7-3D29 cell supernatants with and without a DNase treatment showed no significant difference (Fig. 5C). The HBV RNA Roche MWF kit used here is the manual version of the Roche cobas® 6800/8800 automated IA for HBV RNA quantification that does not include a DNase treatment in its workflow.¹⁷,¹⁸ The lack of difference in RNA quantification with or without the DNase treatments validates the use of Huh7-3D29 supernatants as an RNA standard for both the HBV RNA MWF and the cobas® 6800/8800 automated assay.

## Discussion

Circulating HBV RNAs have emerged as a new biomarker that reflects the transcriptional activity of cccDNA¹²,¹³ and may help monitor both CHB infection¹¹,¹⁴,¹⁵ and the antiviral effect of new anti-HBV therapeutics. Several in house PCR-based assays for circulating HBV RNAs have been described¹³ and two investigational RUO assays have been developed by industry.¹⁶⁻¹⁸ These assays use different DNA (e.g., the WHO HBV DNA standard¹⁹⁻²⁴) or RNA (e.g., synthetic arRNAs⁵⁴) to calibrate circulating HBV RNA quantification.¹⁷,¹⁸ The use of HBV DNA as a standard has been challenged²⁵ and arRNA synthesis is unpractical and costly for routine use. We describe here the development and characterization of an HBV RNA standard suitable for all PCR-based HBV RNA quantification assays. From a theoretical point of view, an HBV RNA standard could be produced by: i) extracting HBV RNA from patients' sera/plasma; ii) in vitro transcription of pregenomic or sub-genomic HBV RNAs; in both cases the RNA would be naked (e.g. not in viral particles, capsids or extracellular vesicles/exosomes) and neither approach would allow for standardization of the extraction step; if sub-genomic RNAs are used, the standard may not be applicable to all HBV RNA detection strategies, especially if PCR target sequences are located in the 5' region of the 3.5 kb RNAs; iii) engineering HBV cell lines to exclusively, or predominantly, produce RNA-containing viral particles similar to those found in patients' sera. These different

approaches must balance quantitative yield, convenient production setting and costs with the need to avoid, or at least minimize, the impact of residual HBV DNA on the HBV RNA assay. Here, we have chosen to generate a stable cell line carrying an integrated HBV genome with mutations that would allow pgRNA encapsidation but prevent its reverse transcription into rcDNA due to the expression of a non-functional viral polymerase. Three candidate mutations (YMAA, Y63F and A1G) have been selected according to their expected impact on pgRNA encapsidation, reverse transcription, rcDNA formation, global viral production³⁵⁻⁴³ and their capability, alone or in combination, to result in an inversion of the HBV DNA/RNA ratio with a higher proportion of pgRNA than rcDNA in cell supernatants.

We have down-selected the best Huh7-derived clonal cell line carrying the double mutation in the catalytic site of the polymerase (YMAA mutation) and the TP domain (Y63F) of the polymerase (Huh7-3D29) that displays an inversion of the secreted HBV DNA/RNA ratio and maintains the desired RNA secretory phenotype over several freeze-thaw and amplification cycles with minimal residual HBV DNA secretion. If one considers the performing characteristics of either Roche MWF assay used in this study or the related HBV RNA cobas® 6800/8800 automated investigational assay,¹⁷,¹⁸ both tolerating ~$10^6$ DNA:RNA ratios without losing RNA specificity and linearity, the HBV RNA standard produced and secreted by the Huh7-3D29 cell line fulfill this criterion. Notably, HBV RNA quantification of Huh7-3D29 supernatants with the Roche MWF showed no significant difference with and without DNase treatment, supporting the use of the Huh7-3D29-derived HBV RNA standard with both automated and manual assays. Additional observations validate the use of Huh7-3D29 cell supernatants as an HBV RNA standard. First, the quantification of HBV RNAs with the Roche MWF of standard curves obtained from 10-fold dilution of Huh7-3D29 supernatants and a synthetic HBV arRNA showed the same profile. Second, the CTs obtained with both standards showed no significant differences. More importantly, the results of iodixanol/sucrose gradients and 5'RACE coupled with single molecule long-read Nanopore sequencing indicate that, as observed in patients with CHB, the majority of secreted HBV RNAs are full length or spliced 3.5 kb species in naked capsid or virion-like particles. These results support the relevance of the Huh7-3D29 supernatants as a standard to quantify HBV RNAs with investigational RUO assays and in house PCR-based HBV RNA assays.

The requirements for a WHO IS is a lyophilized preparation with proven long-term stability that is available in adequate quantities to last several years.²² All viral nucleic acid WHO ISs have been obtained from serum preparation. Therefore, the production of an international HBV RNA standard from a stable clonal cell line would be a novelty and would guarantee sustained access. The Huh7-3D29 cell line is deposited in the CNCM repository (https://www.pasteur.fr/fr/sante-publique/centre-ressources-biologiques/collection-nationale-culturesmicro-organismes-cncm) and constitutes a potentially unlimited source of HBV RNA standard. Twenty very early passage vials of Huh7-3D29 are conserved in liquid nitrogen at the INSERM Lyon CAT Biobank DC-2008-235 (Ethical Committee CPP 11/040) to guarantee a steady supply of the HBV RNA standard to support its validation and diffusion. One 175 cm² flask of Huh7-3D29 cell provides, in 9
A

Armored RNA (Roche)

<figure><img src="image_12.png" /></figure>

<table><thead><tr><td>Ct of diluted arRNA</td><td>Copies/ml</td><td>Ct of diluted 3D29 RNA</td></tr></thead><tbody><tr><td>19.52</td><td>10<sup>5</sup></td><td>18.70</td></tr><tr><td>22.59</td><td>10<sup>4</sup></td><td>22.80</td></tr><tr><td>26.44</td><td>10<sup>3</sup></td><td>26.07</td></tr><tr><td>29.38</td><td>10<sup>2</sup></td><td>29.96</td></tr><tr><td>32.05</td><td>10<sup>1</sup></td><td>34.09</td></tr></tbody></table>

3D29 HBV RNA

<figure><img src="image_13.png" /></figure>

B

Huh7-3D29 p8

<figure><img src="image_14.png" /></figure>

Huh7-3D29 p11

<figure><img src="image_15.png" /></figure>

C

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**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.</figcaption></figure>

days, enough material for 1,300 standard curves and the production can easily be scaled up. As a comparison, the HBV nucleic acid WHO IS produced 2,000 aliquots of 0.5 ml with a concentration of $1 \times 10^6$ IU/ml and the second, third and fourth HBV WHO standard productions were in the same range.¹⁹,²⁰,²² The next steps for developing the Huh7-3D29

HBV RNA standard into an internationally established standard or a WHO-endorsed standard will include i) the lyophilization of the standard material, ii) the evaluation of its performance by a large panel of laboratories, iii) the analysis of its performance by the independent expert committee on biological standardization of the WHO.²¹
Altogether, the establishment of the Huh7-3D29 cell line with a continuous production of HBV RNA-containing supernatants will allow us to share this viral RNA material with

research and diagnostic laboratories to standardize HBV RNA quantification and assist the evaluation of emerging HBV cure strategies.

## Affiliations

¹IHU Lyon, Lyon Hepatology Institute, Lyon, France; ²Cancer Research Center of Lyon (CRCL), UMR Inserm U1052 / CNRS 5286, Lyon, France; ³University of Lyon, University Claude Bernard Lyon 1, 69008 Lyon, France; ⁴Department of Hepatology, Hospices Civils de Lyon, France; ⁵Laboratoire de Virologie, Institut des Agents Infectieux, Hospices Civils de Lyon, Lyon, France; ⁶Department of Internal Medicine, SCIAC and the IIT Center for Life Nanoscience, Sapienza University, Rome, Italy

## Abbreviations

arRNA, armored RNA; cccDNA, covalently closed circular DNA; CHB, chronic hepatitis B; ddPCR, droplet digital PCR; HBsAg, hepatitis B surface antigen; IA, investigational assays; IFN, interferon; IS, International Standard; MWF, Manual Workflow; NMEs, new molecular entities; NUCs, nucleos(t)ide analogues; pgRNA, pregenomic RNA; 5'RACE, 5' rapid amplification of cDNA ends; RUO, research use only; sEVs, small extracellular vesicles; WHO, World Health Organization; WT, wild-type.

## Financial support

This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the second "Investissements d'Avenir" program (reference: ANR-17-RHUS-0003).

## Conflict of interest

The authors disclose no conflicts.
Please refer to the accompanying ICMJE disclosure forms for further details.

## Authors' contributions

Conceptualization: ML and FZ; Funding acquisition: ML and FZ; Formal Analysis: AP, XG, MLP, BT; Investigation: AP, FCT, DB, MLP, HT, FB, CS, BT; Methodology: AP, DB, XG; Resources: MLP, ML, FZ; Supervision: ML, BT, FZ; Visualization: AP, ML; Writing – original draft: AP, ML; Writing – review & editing: all authors.

## Data availability statement

Data are available at reasonable request to the corresponding author.

## Acknowledgements

Graphical abstract created with BioRender.com.

## Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhepr.2024.101124.

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

Received 19 July 2023; received in revised form 16 May 2024; accepted 23 May 2024; Available online 25 May 2024