Development of hepatitis B virus (HBV) serum RNA biomarker assay as a surrogate measure of intra-hepatic HBV replication

Abstract

Background: Over 296 million people worldwide are living with chronic hepatitis B (CHB) infection who require monitoring of viral activity and disease progression. Serum HBV RNA is a promising, although poorly characterized in its encapsidated form, new biomarker in CHB management. No standardized method for serum HBV RNA quantification has been established. Aims: The project aims to characterize the HBV serum transcriptome in order to better understand its composition and to develop and validate, both analytically and clinically, a 3′ RACE RT-qPCR assay for quantification of relevant transcripts within serum HBV RNA. Methods: Nanopore long read sequencing and Northern blotting were employed to characterize the HBV RNA in the serum of 13 patients in different phases of CHB. Sequencing data analysis was done using three isoform detection workflows. A 3′ RACE RT-qPCR method was developed using published primers for the most relevant RNA species determined by serum HBV RNA characterization. The analytical limit of detection and quantification, linearity, inter- and intra-assay repeatability, and clinical specificity and sensitivity were evaluated using synthetic pre-genomic RNA (pgRNA) and specimens from various patient populations. Intra- and inter-laboratory ring trials involving three laboratories were completed. Results: Long read sequencing found higher proportions of spliced variants in patients with HBV genotype B and C, and in HBeAg positive patients with ALT ≤ ULN (upper limit of normal). All chosen isoform detection workflows showed high agreement in most samples, and pgRNA was the most abundant isoform in most patients. Northern blotting was ineffective at detecting serum HBV RNA. The 3′ RACE RT-qPCR assay performed well during analytical and clinical validation and inter- and intra-laboratory analysis demonstrated moderate to high agreement among participants. Conclusions: Long read sequencing is a promising tool for the characterization of the serum HBV transcriptome. The developed 3′ RACE RT-qPCR assay specific for HBV pgRNA, is repeatable, and employs a biologically relevant RNA standard suitable for medium throughput laboratories. Method standardization is required to facilitate the comparison of studies and better understand the clinical role of this novel biomarker.