Transcriptomic Quantitation of Hepatitis B Virus Surface Antigen from Integration

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Project Summary Hepatitis B virus (HBV) surface antigen (HBsAg) is a hallmark in patients with chronic HBV infection (CHB). Given its close association with clinical outcomes in CHB patients, HBsAg seroclearance is required in achieving an HBV functional or complete cure. Toward this direction, a major barrier is relevant to its biological sources. Besides a nuclear reservoir of covalently closed circular DNA (cccDNA), HBsAg could also be produced from cellular integration. Currently, the contribution of HBV integration to HBsAg remains elusive due to the lack of a method for quantitative measurement. Transcription from integrated HBV DNA has several characteristics, such as the retention of human sequences, an early termination, and the interruption of HBV Core antigen. These features have been used to infer the relative abundance of integration-derived HBsAg transcripts by qRT-PCR, digital droplet PCR (ddPCR), and next-generation sequencing (NGS). However, these approaches require liver tissue and have limited use with blood samples from which ~50% of HBeAg- negative patients have total HBV RNA below the lower limit of quantification of qRT-PCR. Bait-based target enrichment in NGS could enhance the sensitivity but extensive microhomology between HBV and human genomes lowers the efficiency. To address these issues, we have developed a novel method through multiple technical advances from our lab, including template-dependent multiple displacement amplification (tdMDA) (BioTechniques 2017), a novel target enrichment strategy via 7‑deaza‑dGTP‑resistant enzymatic digestion (TEED) (BMC Res Notes 2020, patented), and a read simulation to evaluate relative abundance among reference genomes (J Virol Methods 2022). The combination of tdMDA, TEED, and the read simulation, termed MATESim, was applied to five CHB patient serum samples. Integration-derived transcripts accounted for variable portions of total HBsAg transcripts, ranging from 1.8% to 94.3%. These results support the hypothesis that MATESim would be a noninvasive method to quantitate HBsAg from integration at the level of transcription. This hypothesis will be evaluated using patient specimens from the Hepatitis B Research Network (HBRN). First, we will validate MATESim by studying paired serum/liver samples. HBsAg transcripts from cccDNA and integrated HBV DNA may have different mechanisms for release to circulation. It is unknown whether their relative abundance determined by MATESim in circulation is a recapitulation, an underestimation, or an overestimation of liver data. This knowledge will help to interpret data from circulation by MATESim. Second, we will study serum samples from 59 patients with defined HBV phenotypes in the HBRN cohort. The experiment will allow us to gain insights into the magnitude and between-patient variance of integration-derived HBsAg in this patient cohort at a 95% confidence interval. Taken together, as a non- invasive method to quantitate integration-derived HBsAg, MATESim would be a turning point in translational research, clinical trials, and patient management for the globally 257 million people infected with HBV.